Modified vaccinia ankara expressing p53 in cancer immunotherapy

ABSTRACT

Mutations to the tumor suppressor protein p53 have been observed in 40-60% of all human cancers. These mutations are often associated with high nuclear and cytoplasmic concentrations of p53. Since many tumors exhibit highly elevated p53 levels, the protein is an attractive target for cancer immunotherapy. Unfortunately, p53 is an autoantigen that is likely to be tolerated as a self-protein by the immune system. The present invention is based on the discovery that this self-tolerance can be overcome by administration of recombinant modified vaccinia Ankara (MVA) containing a nucleic acid that encodes p53 (rMVAp53). The invention discloses a method of generating a p53-specific CTL-response to tumor cells expressing mutated p53 by administering a composition comprising rMVAp53. Administration of rMVAp53 decreases tumor development, tumor growth, and mortality in a variety of malignant cell types. These effects are enhanced by administration of CTLA-4 blocker and/or CpG oligodeoxynucleotide immunomodulators.

GOVERNMENT INTEREST

[0001] This invention was made with government support in part by grantsfrom the NIH, Division of AIDS (RO1-Al43267 and R21-Al44313) and NCI:RO1:CA77544, PO1-CA30206, R29-CA70819, and CA33572. The government mayhave certain rights in this invention.

BACKGROUND OF INVENTION

[0002] The present utility application claims priority to provisionalpatent application U.S. Ser. No. 06/436,268 (Ellenhorn and Diamond),filed Dec. 23, 2002, and U.S. Ser. No. 06/466,607 (Ellenhorn andDiamond), filed Apr. 30, 2003, the disclosures of which are incorporatedby reference in their entirety herein.

FIELD OF THE INVENTION

[0003] The present invention relates to the fields of virology,molecular biology, and tumor immunology. Specifically, this inventionrelates to compositions and methods for eliciting immune responseseffective against malignancies expressing p53.

BACKGROUND

[0004] p53 is a tumor suppressor protein that regulates the expressionof certain genes required for cell cycle arrest or apoptosis. The tumorsuppressor-gene encoding p53 is activated by DNA damage, cell stress, orthe aberrant expression of certain oncogenes (Levine 1997). Onceactivated, wild type p53 (wt p53) serves to temporarily arrest the cellcycle, allowing time for DNA repair and preventing cells with damagedDNA from proliferating uncontrollably (Levine 1997). p53 is alsoinvolved in inducing apoptosis in cells with certain types ofphysiologic damage (Levine 1997).

[0005] Mutations in p53 that functionally inactivate its growthsuppressing ability have been observed in 40-60% of all human cancers,and are associated with the malignant phenotype (Hainaut 2000).Mutations to p53 occur as early events in tumorigenesis (Millikan 1995;Querzoli 1998; Allred 1993), abrogating the ability of the protein tosuppress cell division (Finlay 1989; Eliyahu 1989). The regulation ofp53 expression in cells can occur at the level of p53 mRNA abundance orat the level of p53 protein abundance. Mutations of p53 are oftenassociated with high nuclear and cytoplasmic concentrations of the p53protein, due to the prolonged half-life of the mutated protein. Manytumors are characterized by highly elevated intracellular p53 levelscompared to nonmalignant cells. Other tumors synthesize large amount ofmutated p53, but contain low or below normal steady-state levels ofintracellular p53, presumably as a result of accelerated intracellulardegradation of the protein. Overexpression of p53 is an independentpredictor of more aggressive cancers (Turner 2000; Elkhuizen 2000;Zellars 2000), lymph node metastases (Pratap 1998), failure to respondto standard therapies (Berns 1998; Berns 2000), and mortality (Sirvent2001; Querzoli 2001).

[0006] Missense point mutations are the most frequent p53 mutations incancer, leaving the majority of the p53 protein in its wild type form(wt p53). Although p53 mutations may represent true tumor specificantigens, most of these mutations occur at sites that do not correspondto immunologic epitopes recognized by T cells (Wiedenfeld 1994). Becauseof this, any widely applicable p53-directed immunotherapy must target wtp53. In experimental models, it has been possible to target p53 becausethe mutated molecule is associated with high nuclear and cytoplasmicconcentrations of the p53 protein (Finlay 1988). p53 is an attractivetarget for adaptive immune response because the intracellularconcentration of nonmutated p53 in healthy cells is very low (Zambetti1993; Reich 1984). This means that healthy cells expressing non-mutantp53 will most likely escape an enhanced immune response toover-expressed mutant p53 (Offringa 2000).

[0007] p53, like most tumor associated antigens that are recognizable bythe cellular arm of the immune system, is an autoantigen (Rosenberg2001). The fact that p53 is an autoantigen widely expressed throughoutdevelopment (Schmid 1991), coupled with the fact that the majority ofmutated p53 being expressed in tumors has the same structure as the wildtype protein, means that tumor-expressed p53 is likely to be toleratedas a self-protein by the immune system. This tolerance, which has beenshown by functional and tetramer studies in mice to exist at thecytotoxic T lymphocyte level (CTL) (Theobald 1997; Erdile 2000), limitsthe effectiveness of p53-directed immunotherapies. To be successful, aneffective immunotherapy must overcome this tolerance without alsoinducing autoimmunity against normal cells and tissues (Theobald 1997;Erdile 2000; Hernandez 2000). Small numbers of self-reacting T cellsescape during the processes involved in the immune tolerance.

[0008] Tumors overexpressing p53 have been eliminated in murine modelsby the systemic administration of epitope specific CTL (Vierboom 2000a;Vierboom 2000b; Vierboom 1997; Hilburger 2001), epitope pulsed dendriticcells (DC) (Mayordomo 1996), or mutant p53 epitope with IL-12(Noguchi-1995). Each of these strategies has considerable drawbacks withregards to clinical applicability. CTL infusion and infusion of epitopepulsed dendritic cells are time consuming and expensive, because theisolation, culturing, and reinfusion of cells must be performed individually for each patient. Conversely, in order to produce any effect,the cell-free vaccination strategies previously used required eitherintratumoral injections or vaccination prior to tumor challenge, neitherof which represents a practical approach in the clinical setting. Thereis thus a need for simplified, efficient, and widely applicableimmunotherapeutic strategies in the treatment of cancer.

SUMMARY OF THE INVENTION

[0009] The p53 gene product is overexpressed in a majority of cancers,making it an ideal target for cancer immunotherapy. The efficacy ofthese therapies has been limited, however, by the fact thattumor-expressed p53 is likely to be tolerated as a self-protein by theimmune system. The present invention is based on the discovery that thisself-tolerance can be overcome by administration of recombinant MVAcontaining a nucleic acid that encodes p53 (rMVAp53). Administration ofp53 is shown to greatly decrease tumor development, tumor growth, andmortality in mice challenged with a variety of malignant cell types. Itis also shown that the therapeutic effects of rMVAp53 are enhanced byadministration of a CTLA4 blocker or CpG oligodeoxynucleotide (CpG ODN)immunomodulator. This enhancement is greatest when both immunomodulatorsare administered. The present invention provides a recombinant MVAcomposition for use in the treatment of cancer, a method of treatingcancer using this composition, and a kit for administration of thecomposition.

[0010] In a first aspect, the present invention provides a compositioncomprising recombinant MVA that contains a nucleic acid encoding p53.Preferably, the p53 encoded by the recombinant MVA is wt human p53.According to the present invention, the composition may also contain aCTLA-4 blocker and/or a CpG ODN.

[0011] In another aspect, the present invention provides a method fortreating a subject having a p53-expressing malignancy. This method isbased on administration of a recombinant MVA containing a nucleic acidthat encodes p53. Preferably, the method also calls for administrationof a CTLA-4 blocker and/or CpG ODN as an immunomodulator. In a thirdaspect, the present invention provides a kit for treating ap53-expressing malignancy. This kit contains a recombinant MVAcontaining a nucleic acid that encodes p53, and may also contain a CTLA4blocker and/or CpG ODN as an immunomodulator. In a final aspect, thisinvention provides for an MVA recombination plasmid containing a nucleicacid insert that encodes wt human p53.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1: PCR analysis of the pLW22-hup53 construct. rMVAhup53injected (lanes 1, 2) and wtMVA infected (lanes 3, 4) BHK cells weresubjected to total DNA extraction and PCR amplification using wtMVA(lanes 2, 4) or hup53 (lanes 1, 3) specific primers. The rMVAhup53product was shown to have no contaminating wtMVA.

[0013]FIG. 2: Expression of mup53 by cells infected with rMVAmup53.Cells infected with rMVAmup53 express mup53 at high levels, confirmingthat MVA is a suitable vaccine vector. Cell lysates were subjected toSDS-PAGE and Western blotting. The lanes are designated as follows: 1)Meth A, unmanipulated. Meth A sarcoma cells, 2) HCMV IE1 exon4-rMVAinfected BHK cells, 3-4) rMVAmup53 infected BHK cells (loaded 0.125 ul,0.25ul cell lysates respectively), 5) rAdp53, and 6) rAdpp65 infectedHEK 293 cells. All lanes were loaded with 20 μl sample unless indicatedspecifically.

[0014]FIG. 3: Generation of a p53-specific CTL response by rMVAmup53 invitro. A single intraperitoneal (i.p.) vaccination with rMVAmup53generates p53 specific CTL responses that efficiently kill cellsoverexpressing p53. (a) Splenocytes from mice treated with rMVAmup53were harvested at 14 days and restimulated in vitro for 6 days withrAdp53 infected syngeneic LPS blasts. CTL activity was evaluated in astandard 4-h ⁵¹Cr release assay using rVVp53 (solid line) or rWpp65(dashed line) infected 10.1 cells. (b) Splenocytes from rMVAmup53 (solidline) or rMVApp65 (dashed line) vaccinated mice were harvested at 14days following vaccination and restimulated in vitro for 6 days withrAdp53 infected syngeneic LPS blasts. Cytotoxicity was measured againstrVVp53 infected 10.1 cells. (c) Splenocytes harvested 14 days afterrMVAmup53 (solid line) or rMVApp65 (dashed line) vaccination wererestimulated in vitro for 6 days using syngeneic LPS blasts infectedwith rMVAp53. Cytotoxicity was measured against Meth A cells by astandard 4-h ⁵¹Cr release assay.

[0015]FIG. 4: Effect of vaccination with rMVAmup53 on Meth A tumorprevention. Balb/c mice were injected subcutaneously (s.c.) with 5×10Meth A cells. On day 5, mice were vaccinated with either 5×10⁷ pfu ofrMVAmup53 (MVAp53) (n=16), 5×10⁷ pfu rMVApp65 (MVApp65) (n=16), or PBS(n=12). The survival plot shows the proportion of surviving animals ineach group as a function of days post tumor challenge. The improvementof the mice vaccinated with rMVAmup53 over both control groups isstatistically significant (P<1) as determined by the log rank test.

[0016]FIG. 5: Effect of vaccination with rMVAmup53 plus anti-CTLA4 mAbon established Meth A tumors. Mice were injected s.c. with a rapidlylethal dose of 10⁶ Meth A cells. On days 6, 9, and 12 mice were injectedi.p. with either anti-CTLA-4 mAb (CTLA4 mAb) or control mAb. On day 7,mice were vaccinated with either 5×1 pfu rMVAp53 (MVAp53) or 5×1 pfurMVApp65 (MVAapp65). The survival plot shows the proportion of survivinganimals in each group. The survival advantage of mice vaccinated withrMVAp53 plus anti-CTLA4 mAb (n=14) over control animals receivingrMVApp65 plus CTLA-4 (n=14), rMVAp53 plus control ab (n=14), or rMVApp65plus control ab (n=6) is statistically significant (P<0.001) asdetermined by the log rank test.

[0017]FIG. 6: Effect of vaccination with rMVAmup53 plus anti-CTLA-4 mAbon established 11A-1 tumors. Balb/c mice were injected s.c. with 2×10⁶11A-1 cells (p=0.00044, comparing rMVAmup53 plus anti-CTLA-4 mAb to allother groups). Anti-CTLA-4 mAb 9H10 (CTLA4 mAb) or the control hamsterisotype matched polyclonal antibody (isotype matched Ab) were injectedi.p. on days 4, 7, and 10 at 100, 50, and 50 μg dose, respectively. Onday 5, mice were vaccinated i.p. with either 5×10⁷ pfu of rMVAmup53(MVAp53), 5×10⁷ pfu rMVApp65 (MVApp65), or PBS. Each line represents themean and standard deviation of eight mice.

[0018]FIG. 7: Effect of vaccination with rMVAmup53 plus anti-CTLA-4 mAbon established MC-38 tumors. C57BL/6 mice were injected s.c. with 1×10⁶MC-38 cells (p=0.0001, comparing rMVAmup53 plus anti-CTLA-4 mAb to allother groups). Anti-CTLA-4 mAb 9H10 (CTLA4 mAb) or the control hamsterisotype matched polyclonal antibody (isotype matched Ab) were injectedi.p. on days. 4, 7, and 10 at 100, 50, and 50 μg dose, respectively. Onday 5, mice were vaccinated i.p. with either 5×10⁷ pfu of rMVAmup53(MVAp53), 5×10⁷ pfu rMVApp65 (MVApp65), or PBS. Each line represents themean and standard deviation of eight mice.

[0019]FIG. 8: Effect of vaccination with rMVAmup53 plus CpG ODN onestablished 11A-1 tumors. Balb/c mice were injected s.c. with 2×10⁶11A-1 cells (p 0.00002, comparing rMVAmup53 plus CpG ODN to all othergroups). 15 nmoles of CpG ODN (CpG) was injected i.p. on days 4, 9, and14. On day 5, the mice were immunized i.p. with either 5×10⁷ pfu ofrMVAmup53 (MVAp53), 5×10⁷ pfu of rMVApp65 (MVApp65), or PBS.

[0020]FIG. 9: Effect of vaccination with rMVAmup53 plus CpG ODN onestablished Meth A tumors. Balb/c mice were injected s.c. with 1×10⁶Meth A cells (p=0.0015, comparing rMVAmup53 plus CpG ODN to all othergroups). 15 nmoles of CpG ODN (CpG) was injected i.p. on days 4, 9, and14. On day 5, the mice were immunized i.p. with either 5×10⁷ pfu ofrMVAmup53 (MVAp53), 5×10⁷ pfu of rMVApp65 (MVApp65), or PBS.

[0021]FIG. 10: Effect of vaccination with rMVAmup53 plus CpG ODN onestablished MC-38 tumors. C57BL/6 mice were injected with 1×10⁶ MC-38cells (p=0.0004, comparing rMVAmup53 plus CpG ODN to all other groups).15 nmoles of CpG ODN (CpG) was injected i.p. on days 4, 9, and 14. Onday 5, the mice were immunized i.p. with either 5×10⁷ pfu of rMVAmup53(MVAp53), 5×10⁷ pfu of rMVApp65 (MVApp65), or PBS.

[0022]FIG. 11: Effect of vaccination with rMVAmup53 plus anti-CTLA-4 mAband CpG ODN on established 11A-1 tumors. Balb/c mice (n=8) were injecteds.c. with 2×10⁶11A-1 cells. Anti-CTLA-4 mAb (CTLA4 mAb) was injectedi.p. on days 14, 17, and 20 at 100, 50, and 50 μg dose, respectively. 15nmoles of CpG ODN (CpG) was injected i.p. on days 14, 19, and 24. On day15, mice were vaccinated i.p. with either 5×10⁷ pfu of rMVAmup53(MVAp53), 5×10⁷ pfu rMVApp65, or PBS. The survival plot shows theproportion of surviving animals in each group as a function of days posttumor challenge. p=0.02 comparing combined CpG ODN and anti-CTLA-4 mAbto CpG ODN alone, and p=0.01 comparing combined CpG ODN and anti-CTLA-4mAb to anti-CTLA-4 mAb alone.

[0023]FIG. 12: Effect of vaccination with rMVAmup53 plus anti-CTLA-4 mAband CpG ODN on established MC-38 tumors. C57BL/6 mice (n=8) wereinjected s.c. with 1×10⁶ MC-38 cells. Anti-CTLA-4 mAb was injected i.p.on days 4, 7, and 10 at 100, 50, and 50 μg dose, respectively. 15 nmolesof CpG ODN was injected i.p. on days 4, 9, and 14. On day 5, mice werevaccinated i.p. with either 5×10⁷ pfu rMVAmup53, 5×10⁷ pfu MVApp65, orPBS. The survival plot shows the proportion of surviving animals in eachgroup as a function of days post tumor challenge. p=0.002 comparingcombined CpG ODN and anti-CTLA-4 mAb to CpG alone, and p=0.001 comparingcombined CpG ODN and anti-CTLA-4 mAb with anti-CTLA-4 mAb alone.

[0024]FIG. 13: Cellular requirements for anti-CTLA-4 mAb immunomodulatoreffect on Meth A tumors. Balb/c mice (a) or IFN-γ^(KO) Balb/c mice (b)were injected s.c. with a rapidly lethal dose of 10⁶ Meth A cells.Groups of mice from both populations were injected i.p. with depletingdoses of anti-CD4, anti-CD8, anti-NK1.1, or control mAb on days −1, 1,3, and 10, and weekly thereafter. On days 6, 9, and 12 mice wereinjected i.p. with either anti-CTLA-4 mAb (CTLA4 mAb) or control mAb. Onday 7, mice were vaccinated with either 5×10⁷ pfu rMVAp53 (MVAp53) or5×10⁷ pfu rMVApp65 (MVAapp65). (a) Mean tumor growth was calculated foreach group of Balb/c mice, with error bars illustrating standarddeviation. The last datapoint for each line represents the firstmortality. (b) The proportion of surviving IFN-γ^(KO) Balb/c mice isplotted.

[0025]FIG. 14: Cellular requirements for CpG ODN immunomodulator effectson 11A-1 tumors. Balb/c mice were injected s.c. with 2×10⁶ 11A-1 cells.15 nmoles of CpG ODN was injected i.p. on days 4, 9, and 14. On day 5,mice were vaccinated i.p. with 5×10⁷ pfu of rMVAmup53. Mice wereinjected i.p. with depleting doses of anti-CD4 (CD4), anti-CD8 (CD8),anti-NK1.1 (NK), or control mAb on days 4, 6, 8, and 15, and every 7days thereafter. Tumors were measured twice weekly in three dimensions.p=0.004 by two-sided Wilcoxon test, comparing CD8⁺ depleted to all othergroups. p=0.007, comparing anti-NK1.1 to anti-CD4 and control mAb.

[0026]FIG. 15: Cellular requirements for anti-CTLA-4 mAb immunomodulatoreffects on 11A-1 tumors. Mice were injected s.c. with 2×10⁶ 11A-1 cells.Anti-CTLA-4 mAb was injected i.p. on days 4, 7, and 10 at 100, 50, and50 μg/dose, respectively. On day 5, the mice were vaccinated i.p. with5×10⁷ pfu rMVAmup53. The mice were depleted of CD84, CD44, or NK cellsby i.p. injection with the relevant mAb or control mAb on days 4, 6, 8,and 15, and then every 7 days thereafter. Tumors were measured twiceweekly in three dimensions with calipers. Each curve represents the meanand standard deviation of 8 mice. p=0.004, comparing CD8⁺ depleted toall other groups. p=0.008, comparing CD4⁺ depleted to NK depleted andcontrol groups.

[0027]FIG. 16: Contribution of TLR 9 to the CpG ODN immunomodulatoreffect. TLR9^(−/−) (p=0.0009, comparing anti-CTLA-4 mAb to CpG ODNgroup) mutant C57BL/6 mice were injected s.c. with 1×10⁶ MC-38 cells.Mice were treated with anti-CTLA-4 mAb (CTLA4 mAb) on days 4, 7, and 10at 100, 50, and 50 μg/dose, respectively, or with 15 nmoles of CpG ODNon days 4, 9, and 14. On day 5, all mice were vaccinated i.p. with 5×10⁷pfu of rMVAmup53. Tumors were measured twice weekly in three dimensionswith calipers. Each curve represents the mean and standard deviation of8 mice.

[0028]FIG. 17: Contribution of IL-6 to the CpG ODN immunomodulatoreffect. IL-6⁺ (p=0.02, comparing anti-CTLA-4 mAb to CpG ODN group byWilcoxon 2-sided RankSum Test) mutant C57BL/6 mice were injected s.c.with 1×10⁶ MC-38 cells. Mice were treated with anti-CTLA-4 mAb (CTLA4mAb) on days 4, 7, and 10 at 100, 50, and 50 μg/dose, respectively, orwith 15 nmoles of CpG ODN on days 4, 9, and 14. On day 5, all mice werevaccinated i.p. with 5×10⁷ pfu of rMVAmup53. Tumors were measured twiceweekly in three dimensions with calipers. Each curve represents the meanand standard deviation of 8 mice.

[0029]FIG. 18: Expression of hup53 by cells infected with rMVAhup53. BHKcells were injected with purified rMVAhup53 (MVA/p53). Expression ofhup53 was measured at 24 and 48 hours. Cell lysates were subjected toSDS-PAGE and Western blotting. Lane 1: BHK cells injected with controlMVA; Lane 2: BHK cells infected with rMVAhup53 for 24 hours; Lane 3: BHKcells infected with rMVAhup53 for 48 hours. All lanes were loaded with20 μl of sample.

[0030]FIG. 19: Effect of vaccination with rMVAhup53 plus anti-CTLA-4 mAband CpG ODN on established 4T1/hup53 tumors. Mice were injected s.c.with 5×10⁴ 4T1/hup53, then vaccinated i.p. with 10⁷ pfu rMVAhup53 or PBScontrol on day 6. On day 16, mice received an rMVAhup53 or PBS boosterinjection, along with 1-5 nmole of CpG ODN and 50 μg of anti-CTLA4 mAb.rMVAhup53 vaccinated mice displayed a significant improvement insurvival (p<0.05, two sided T-test) compared to PBS controls.

DETAILED DESCRIPTION

[0031] The present invention is based on the discovery thatself-tolerance to a protein expressed in both normal and cancerous cellscan be overcome, and that a strong anti-tumor immune response can begenerated without the requirement for intratumoral administration andwithout the production of systemic toxicity or auto-immunity. Theinvention provides novel cell-free compositions and methods for thegeneration of effective immune responses against a wide variety of humanmalignancies, independent of the subject's haplotype or genotype. Theexamples discussed below demonstrate that vaccination with a modifiedvaccinia Ankara vector engineered to express either wild type murine orwild type human p53 (rMVAmup53 or rMVAhup53) stimulates a vigorousp53-specific CTL response. This response can be enhanced by theco-administration of an immunomodulator consisting of a CTLA-4 blockerand/or CpG ODN.

[0032] MVA virus (GenBank Accession Number U94848) is a variant of theAnkara strain of vaccinia virus that was derived by over 570 serialpassages on primary chicken embryo fibroblast. Several properties of MVAas an attenuated poxvirus make it ideal for the generation of atherapeutic response to tumors expressing p53. One advantage of MVA isthat it is able to efficiently replicate its DNA in mammalian cells, yetit is avirulent and does not propagate. This trait is the result oflosing two important host range genes among at least 25 additionalmutations and deletions that occurred during its passages throughchicken embryo fibroblasts (Meyer 1991; Antoine 1998). In contrast toNYVAC (attenuated Copenhagen strain) and ALVAC (host range restrictedavipox), both-early and late transcription in MVA are unimpaired,allowing for continuous gene expression throughout the viral life cycle(Carroll 1997a; Carroll 1997b; Blanchard 1998; Sutter 1992). MVA hasbeen found to be more immunogenic than the Western Reserve (WR) strain,and can be used in conditions of pre-existing poxvirus immunity (Ramirez2000a; Ramirez 2000b). The favorable clinical profile of MVA as arecombinant vaccine delivery vehicle is buttressed by its benign safetyprofile as a smallpox vaccine in Europe in the late 1970's (Mayr 1999;Mayr 1978). MVA was administered to over 120,000 high-risk individuals,including the aged and very young, without serious side effects (Mayr1978). More recently, MVA has also been administered toimmunocompromised non-human primates without adverse outcome (Stittelaar2001). This is in stark contrast to other vectors, such as retrovirusesand adenoviruses, which pose documented risks to the human host.Immunotoxicity of the vector, adjuvant, or immunomodulator used is aparticular point of concern in the immunotherapy of cancer, as mostcancer patients are severely immunocompromised due to chemotherapy,radiation, or the immunosuppressive effects of the cancer itself. MVAwas first developed into a vaccine vehicle in the early 1990's, after itbecame clear that non-attenuated poxviruses such as the WR strain couldnot be safely administered to immunocompromised individuals (Redfield1987; Collier 1991). In summary, the potency of MVA as an expressionvector combined with its safety profile in primates and humans make ithighly attractive as a delivery system for cancer genes.

[0033] Construction of rMVAmup53 and rMVAhup53 is achieved byrecombinant DNA techniques that are well known in the art (Sambrook etal., Molecular Cloning, Cold Spring Harbor Laboratory, 2001; Ausubel etal., Current Protocols in Molecular Biology, John Wiley & Sons, 1986 and2000). The coding sequence of wild type p53 can be conveniently obtainedby RT-PCR using p53-specific primers. These primers hybridize to DNA andserve as initiation sites for DNA synthesis. Nucleotide primers aredesigned to bind at separate sites on opposing duplex strains, therebydefining the intervening sequence as the portion to be amplified.Nucleic acid molecules to be employed as primers will generally includeat least a 10 base pair sequence complementary to the DNA segment to beamplified. Primer selection is well known to those of skill in the art.Primers for the amplification of wt mup53 or wt hup53 can be designed tocontain appropriate restriction sites for subcloning into a suitable MVArecombination plasmid, such as pMCO3, pLW22, pLW51, PUCII LZ or otherMVA transfer vectors well known in the art. The recombination plasmidcontains sequences necessary for expression of the foreign gene insert,as well as the flanking sequences necessary for homologous recombinationinto a chosen-site of deletion in the MVA genome. To generaterecombinant MVA virus, cells are infected with MVA virus and transfectedwith the recombination plasmid containing the foreign gene insert. Afterhomologous recombination between virus and plasmid is allowed to occur,recombinant MVA expressing the inserted gene is isolated.

[0034] Cellular expression of p53 protein following infection withrMVAmup53 or rMVAhup53 was analyzed to determine the fidelity and extentof its expression from recombinant virus. Meth A cells, whichoverexpress mutated p53, were used as a positive control, and HCMV IE1exon4 rMVA infected BHK cells were used as a negative control. Westernblot analysis revealed abundant p53 expression by cells infected withrMVAmup53 or rMVAhup53, as well as by Meth A cells. No detectableexpression of p53 by HCMV IE1 exon 4-rMVA infected BHK cells wasobserved. High levels of p53 expression by rMVAp53 infected BHK cellswas also observed by fluorescence microscopy. The high level of p53expression exhibited by rMVAmup53 and rMVAhup53 compared to other viraland cellular forms demonstrates its usefulness in vaccination protocols.

[0035] In animal experimental models, MVA based vaccines stimulate tumorspecific CTL activity (Espenschied 2003; Drexler 1999) and effectregression of established tumors (Espenschied 2003; Carroll 1997b;Mulryan 2002; Rosales 2000). There are numerous advantages toimmunization with whole protein expressed in MVA. In contrast to peptideimmunization, multiple epitopes can be expressed, and a polyconal hostresponse can be stimulated. Antigen-specific cognate help, which isessential to the propagation of a CTL response, can be achieved throughexpression of a protein in MVA. In addition, expression of whole proteincan result in the stimulation of responses to otherwise crypticepitopes. Immunization with recombinant viruses may also avoid the needfor a complex and expensive approach involving the expansion andadoptive transfer of antigen-specific cells, or the need to generate anindividualized vaccine for a particular cancer patient. This advantageof a recombinant vaccine approach may encourage more widespread clinicaluse to prevent recurrence in patients with earlier stages of disease.

[0036] In vitro experiments were run to determine whether vaccinationwith rMVAmup53 could break p53 tolerance, resulting in the generation ofp53-specific CTL. Splenocytes were harvested from mice following asingle intraperitoneal (i.p.) vaccination with rMVAmup53, andrestimulated in vitro with p53 over-expressing cells. The splenocytesrecognized and lysed wt p53 over-expressing targets. In contrast,splenocytes from mice vaccinated with rMVApp65, which stimmulatesvigorous pp65 specific CTL responses, did not recognize the p53over-expressing targets, demonstrating the specificity of the lymphocyteresponse. rMVAmup53 vaccination can also stimulate CTL recognition ofMeth A cells, which express mutated p53. Restimulated splenocytes frommice vaccinated with rMVAmup53 recognized mutant p53 over-expressingMeth A, whereas control mice vaccinated with rMVApp65 did not.

[0037] Since a single vaccination with rMVAmup53 resulted in enhancedCTL response, there was sufficient justification to examine the effectof rMVAmup53 vaccination on the growth of Meth A tumor cells in vivo.Administration of rMVAmup53 was shown to inhibit the outgrowth of murinesarcoma Meth A, an immunogenic tumor cells line that overexpressesmutant p53. Mice inoculated with a lethal dose of Meth A tumor cells andvaccinated with rMVAmup53 by i.p. injection three days later exhibitedslower tumor growth and higher survival rates than control animals. Amajority of the vaccinated mice failed to develop tumors entirely, andthese mice were resistant to rechallenge with Meth A after 52 days(Espenschied 2003).

[0038] The above results demonstrate the efficacy of a novel rMVAmup53cell-free vaccine at targeting p53 expressed by a malignant tumor.Additional experiments were performed to determine whether this effectcould be enhanced by addition of a CTLA-4 blocker or CpG ODNimmunomodulator. Immunization with vaccinia viral constructs results inthe uptake and presentation of viral proteins by DC (Norbury 2002). Indraining lymph nodes, the DC present antigen to naïve CD8⁺ T cells,resulting in T cell activation and the subsequent propagation of animmune response (Norbury 2002). Immunomodulator experiments weredesigned to determine the feasibility of augmenting the response torMVAp53 by addressing both the initiation of the response and itspropagation.

[0039] One potent strategy for optimizing tumor vaccines involvesmanipulating negative regulation of T cell responsiveness by using amolecule that blocks CTLA-4 engagement with ligand, a phenomenonreferred to as “CTLA-4 blockade.” CTLA-4 is a cell surface receptorfound on T cells. Activation of CTLA-4 leads to inhibition of T cellresponses. CTLA-4 plays a significant role in regulating peripheralT-cell tolerance by interfering with T-cell activation through bothpassive and active mechanisms (Egen 2002). Application of a CTLA-4blocker in combination with cancer vaccines expressing tumor associatedautoantigens can-, in some cases, result in tumor rejection along withbreaking of tolerance, albeit with the concomitant induction ofautoimmunity (Espenschied 2003; Hurwitz 2000; van Elsas 1999). In vitro,CTLA-4 blockade lowers the T-cell activation threshold and removes theattenuating effects of CTLA-4. CTLA-4 blockade also inhibits Treg cellactivity in vivo (Read 2000). When combined with GM-CSF producing tumorcell vaccines, CTLA-4 blockade results in rejection of establishedpoorly immunogenic melanoma, mammary carcinoma, and prostate carcinomagrafts (Hurwitz 1998; Hurwitz 2000; van Elsas 1999). This occurs througha process, which involves breaking tolerance to tumor associatedantigens. CTLA-4 blocking agents are molecules that specifically bind tothe CTLA-4 receptor and interfere with the binding of CTLA-4 to itscounter-receptors. A CTLA-4 blocking agent can be a monoclonal orpolyclonal antibody, a fragment of an antibody, a peptide, a smallorganic molecule, a peptidomimetic, a nucleic acid such as interferingRNA (iRNA) or antisense molecule, an aptamer, or any domains from CTLA-4ligands, including members of the B7 family of CTLA-4 ligands, whereinsaid ligands can be preferably synthesized as recombinant solubleproteins capable of binding CTLA-4 present on immune cells and blockingCTLA-4 function. Anti-CTLA-4 antibodies may be generated by immunizing ahost animal with CTLA-4 protein or with cells expressing CTLA-4.Monoclonal antibodies to CTLA-4 (anti-CTLA-4 mAb) can be produced byconventional techniques, namely fusing a hybridoma cell with a mammalianimmune cell that produces anti-CTLA-4 antibody. Mammalian cells used togenerated anti-CTLA-4 mAb may include rat, mouse, hamster, sheep, orhuman cells. Anti-CTLA-4 mAbs may be purified from hybridoma cellsupernatants or from ascites fluid. Anti-CTLA-4 antibodies may be humanantibodies generated using transgenic animals (Bruggemann 1991; Mendez1997) or human immunoglobulin phage display libraries (Winter 1994).Anti-CTLA-4 antibodies also encompasses chimeric and humanized (or“reshaped”) antibodies. Chimeric antibodies to CTLA-4 may be generatedthrough recombinant methods to contain the CTLA-4 binding domain of anon-human antibody and the constant domain of a human antibody.Humanized antibodies to CTLA-4 may be generated by recombinant methodsto contain only the CDR regions of non-human anti-CTLA-4 antibodiesplaced on a human antibody structural framework (Jones 1986; Low 1986).Individual residues within the non-human region may be substituted withresidues from the human antibody framework. Conversely, individualresidues within the human antibody framework may be substituted withresidues from the non-human antibody (Foote 1992). Such substitutionsmay be used to increase the binding capabilities of the humanizedantibody or to decrease the immune response against the antibody.Humanized antibodies to CTLA-4 can be the product of an animal havingtransgenic human immunoglobulin constant region genes. They can also beengineered by recombinant DNA techniques to substitute the C_(H)1,C_(H)2, C_(H)3, hinge domains, or other domains with the correspondinghuman sequence, by methods known in the art.

[0040] Oligodeoxynucleotides containing unmethylated CpG(cytosine-phosphate-guanine) motifs are potent immunostimulatory agentsthat can enhance vaccine potency (Krieg 2002). Immune activation by CpGODN initiates with specific binding to the Toll-like Receptor-9 (TLR9)in B cells and plasmacytoid dendritic cells (Krieg 2002). TLR9 ligationin DC results in secondary activation of lymphocyte, macrophage,monocyte, natural killer (NK), and T-cell populations through theelaboration of cytokines generating a T_(H)1 cytokine milieu (Krieg2003). This results in increased NK activity, improved antigenpresentation, and T cell help that can augment both humoral andcell-mediated immune responses. In addition, TLR9 ligation results inthe production of IL-6 by DCs, which helps overcome the suppressiveeffect of CD4⁺ CD25⁺ Treg cells (Pasare 2003). Administration of CpG ODNalone has been shown to exert modest anti-tumor effects in a number ofmurine tumor models (Carpentier 1999; Kawarada 2001; Ballas 2001; Baines2003; Sharma 2003). CpG ODN has been shown to be an effective adjuvantfor a variety of experimental tumor vaccines in mice. It is at least aseffective as Freund's adjuvant, but with higher T_(H)1 activity and lesstoxicity (Chu 1997; Weiner 1997). CpG ODN can enhance the effect ofpeptide (Davila 2000; Stern 2002), protein (Kim 2002), DC (Heckelsmiller2002), idiotype (Baral 2003), and GM-CSF secreting tumor cell vaccines(Sandler 2003). The ability of CpG ODN to prime for TH1 responses andstimulation of NK cells probably accounts for the immunomodulatoractivity in these vaccine approaches and in those described below.

[0041] To determine whether administration of a CTLA-4 blocking agent inconjunction with rMVAmup53 vaccination would be beneficial or wouldinduce autoimmune disease, a monoclonal antibody specific to CTLA-4(anti-CTLA-4 mAb) was used. Vaccination with rMVAmup53 and anti-CTLA-4mAb was shown to effect the rejection of established, palpable Meth Atumors. Mice injected with a high dose of Meth A and vaccinated withrMVAmup53 and anti-CTLA-4 mAb (9H10) only after formation of a palpabletumor nodule exhibited complete tumor regression and lasting tumorimmunity. In vivo antibody depletion studies confirmed that thisantitumor effect was primarily CD8⁺, and to a lesser extent CD4⁺,dependent.

[0042] To establish that the above results were not tumor specific,vaccination with rMVAmup53 and a CTLA-4 blocker immunomodulator wasperformed on mice injected with 11A-1 or MC-38 tumor cells. 11A-1 is arapidly growing malignant cell line that is poorly immunogenic. MC-38 isa colon carcinoma cell line. Mice injected with 11A-1 or MC-38 tumorcells and vaccinated 4 days later with rMVAmup53 and anti-CTLA-4 mAbrejected their tumors. Similar results were seen when the anti-CTLA-4mAb was replaced with CpG ODN. The majority of mice treated withrMVAmup53 and CpG ODN did not develop palpable tumors and developedlasting tumor immunity, rejecting a rechallenge at 60 days.

[0043] The potential additive effect of the anti-CTLA-4 mAb and CpG ODNimmunomodulators was examined by administering both immunomodulators inconjunction with rMVAmup53 to 11A-1 injected mice with palpable tumors.Tumor rejection and prolonged survival were observed in the majority ofmice receiving both immunomodulators in conjunction with rMVAmup53. Micethat received only one immunomodulator in conjunction with rMVA, on theother hand, all eventually succumbed to tumor growth. Not only did thecombination of both immunomodulators provide a greater benefit thaneither immunomodulator acting alone, but their combined benefit wasgreater than the simple addition of the effects of the immunomodulators.Similar results were seen in mice bearing MC 38 tumors.

[0044] To determine the efficacy of a recombinant MVA containing a humanp53 sequence, rMVAhup53 was administered to hupki mice injected with4T1(H-2^(d)) cells that had been transfected with human p53. 4T1(H-2^(d)) is a murine breast carcinoma cell line. Mice were vaccinatedwith rMVAhup53 6 days after injection with 4T1 cells, and vaccinatedagain ten days later. During the second vaccination, CpG ODN andanti-CTLA-4 mAb were administered as well. Mice treated with vaccine andboth immunomodulators exhibited a statistically significant improvementin survival.

[0045] The above results demonstrate the efficacy of a novel rMVAmup53or rMVAhup53 cell-free vaccine at eliciting an immune response targetingp53 in a variety of malignant tumor types, as well as the efficacy ofanti-CTLA4 mAb and CpG ODN as immunomodulators to this vaccine.Accordingly, the present invention provides a composition comprising arecombinant MVA virus engineered to express p53 (rMVAp53). The presentinvention further provides an immunotherapeutic method for eliciting animmune response against a wide range of p53-expressing malignancies byadministering rMVAp53

[0046] Introduction of rMVAp53 into a subject can be performed by anyprocedure known to those skilled in the art, and is not dependent on thelocation of tumor nodules for efficacy or safety. Thus, rMVAp53 can beadministered by intravascular, subcutaneous, peritoneal, intramuscular,intradermal or transdermal injection, to name a few possible modes ofdelivery. rMVAp53 can be prepared as a formulation at an effective dosein pharmaceutically acceptable media, such-as normal saline, vegetableoil, mineral oil, PBS, etc. Therapeutic preparations may includephysiologically tolerable liquids, gel or solid carriers, diluents,adjuvants and excipients. Additives may include bactericidal agents,additives that maintain isotonicity (e.g., NaCl, mannitol), additivesthat maintain chemical stability (e.g., buffers, preservatives) andother ingredients. For parenteral administration, the rMVAp53 may beformulated as a solution, suspension, emulsion or lyophilized powder inassociation with a pharmaceutically acceptable parenteral vehicle.Liposomes or non-aqueous vehicles, such as fixed oils, may also be used.The formulation may be sterilized by techniques known in the art.

[0047] The rMVAp53 formulation can be further enhanced with acostimulator, such as a cytokine, tumor antigen, an antigen derived froma pathogen, or any immunomodulator. The costimulator can be any agentthat directly or indirectly stimulates an immune response in combinationwith the rMVAp53, and maybe selected for its ability to modulate APC orT-cell function. For example, MVA can be engineered to express GM-CSF,IL-12, or other stimulatory cytokines to produce a costimulator, and thecombination of rMVAp53 and costimulator (here: MVA expressing thestimulatory cytokine) can be introduced into the subject. The treatmentmay be performed in combination with administration of cytokines thatstimulate antigen presenting cells, such as granulocyte-macrophagecolony stimulating factor (GM-CSF), macrophage colony stimulating factor(M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 3(IL-3), interleukin 12 (IL-12), and others well known in the art. Othercostimulators include cytokine-transduced tumor cells, such as tumorcells transduced with GM-CSF, as well as tumor cells that have beenirradiated and/or treated with a chemotherapeutic agent ex vivo or invivo. Chemotherapeutic or radiotherapeutic agents are further examplesof costimulators. Thus, rMVAp53 can be administered in conjunction witha variety of costimulators known to those of skill in the art.

[0048] The formulation is administered at a dose effective to increasethe response of T cells to antigenic stimulation. The determination ofthe T cell response will vary with the condition that is being treated.Useful measures of T cell activity are proliferation, the release ofcytokines, including, IL-2, IFNγ, TNFα, etc; T cell expression ofmarkers such as CD25 and CD69; and other measures of T cell activity asknown in the art. The dosage of the therapeutic formulation will varywidely, depending upon the stage of the cancer, the frequency ofadministration, the manner or purpose of the administration, theclearance of rMVAp53 from the subject, and other considerations. Thedosage administered will vary depending on known factors, such as thepharmacodynamic characteristics of the particular agent, mode and routeof administration, age, health and weight of the recipient, nature andextent of symptoms, concurrent treatments, frequency of treatment, andeffect desired. The dose may be administered as infrequently as weeklyor biweekly, or fractionated into smaller doses and administered daily,semi-weekly, etc., to maintain an effective dosage level.

[0049] Generally, a daily dosage of active ingredient can be about10⁶-10¹¹ IU (infectious units)/kg of body weight. Dosage forms suitablefor internal administration generally contain from about 10⁶ to 10¹² IUof active ingredient per unit. The active ingredient may vary from 0.5to 95% by weight based on the total-weight of the composition. In somecases it may be desirable to limit the period of treatment due toexcessive T cell proliferation. The limitations will be empiricallydetermined, depending on the response of the patient to therapy, thenumber of T cells in the patient, etc. The number of T cells may bemonitored in a patient by methods known in the art, including stainingwith T cell specific antibodies and flow cytometry.

[0050] In a preferred embodiment of the present invention, rMVAp53 isadministered in conjunction with an immunomodulator, specifically aCTLA4 blocking agent or a CpG ODN. The combined administration ofrMVAp53 and the CTLA-4 blocking agent anti-CTLA-4 mAb is unexpectedlypotent in producing regression of advanced tumors that are rapidlylethal when left untreated. The same is true of the combinedadministration of rMVAp53 and CpG ODN. Potency is even greater when bothimmunomodulators are administered in conjunction with rMVAp53. Inaddition, the anti-CTLA-4 mAb CpG ODN immunomodulators are nontoxic tothe subject, and capable of generating long lasting immunity to lethalchallenges with tumor cells when administered in conjunction withrMVAp53. As is the case with rMVAp53 alone, introduction of rMVAp53 plusanti-CTLA-4 mAb and/or CpG ODN into a subject can be performed by anyprocedure known to those skilled in the art, and is not dependent on thelocation of tumor nodules for efficacy or safety. Thus, rMVAp53,anti-CTLA4 mAb, and CpG ODN can be administered by intravascular,subcutaneous, peritoneal, intramuscular, intradermal or transdermalinjection, to name a few possible modes of delivery. rMVAp53,anti-CTLA-4 mAb, and CpG ODN can be administered together, separately,or sequentially, in any order, by the same route of administration or bydifferent routes. rMVAp53 plus anti-CTLA-4 mAb and/or CpG ODN can beprepared as formulations at an effective dose in pharmaceuticallyacceptable media, for example normal saline, vegetable oil, mineral oil,PBS, etc. Therapeutic preparations may include physiologically tolerableliquids, gel or solid carriers, diluents, adjuvants and excipients.Additives may include bactericidal agents, additives that maintainisotonicity, e.g. NaCl, mannitol; and chemical stability, e.g. buffersand preservatives and other ingredients. rMVAmup53 plus anti-CTLA-4 mAband/or CpG ODN may be administered as a cocktail or as single agents.For parenteral administration, anti-CTLA-4 mAb and CpG ODN may beformulated as a solution, suspension, emulsion or lyophilized powder inassociation with a pharmaceutically acceptable parenteral vehicle.Liposomes or non-aqueous vehicles, such as fixed oils, may also be used.The formulation may be sterilized by techniques as known in the art.

[0051] The rMVAp53 plus anti-CTLA-4 mAb and/or CpG ODN combination canbe further enhanced with a costimulator such as a cytokine, tumorantigen, or antigen derived from a pathogen. A costimulator can be anyagent that directly or indirectly stimulates an immune response incombination with rMVAp53 or in combination with rMVAp53 plus anti-CTLA-4mAb and/or CpG ODN. For example, MVA can be engineered to expressGM-CSF, IL-12, or other stimulatory cytokine to produce a costimulator,and the combination of rMVAp53 and costimulator (here: MVA expressingthe stimulatory cytokine), or rMVAp53 plus anti-CTLA-4 mAb and/or CpGODN and costimulator can be introduced into the subject. The treatmentmay be performed in combination with administration of cytokines thatstimulate antigen presenting cells, such as granulocyte-macrophagecolony stimulating factor (GM-CSF), macrophage colony stimulating factor(M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 3(IL-3), interleukin 12 (IL-12) and others well known in the art. Othercostimulators include cytokine-transduced tumor cells such as tumorcells transduced with GM-CSF, or tumor cells that have been irradiatedand/or treated with a chemotherapeutic agent ex vivo or in vivo.Chemotherapeutic or radiotherapeutic agents are further examples ofcostimulators. Thus, rMVAp53 either alone or in combination withanti-CTLA-4 mAb and/or CpG ODN can be administered in conjunction with avariety of costimulators known to those of skill in the art.

[0052] The dosage of the therapeutic formulation will vary widely,depending upon the stage of the cancer, the frequency of administration,the manner or purpose of the administration, and the clearance ofrMVAp53, anti-CTLA-4 mAb, and CpG ODN from the subject, among otherconsiderations. The dosage administered will vary depending on knownfactors, such as the pharmacodynamic characteristics of the particularagent, mode and route of administration, age, health and weight of therecipient, nature and extent of symptoms, concurrent treatments,frequency of treatment and effect desired. The dose may be administeredas infrequently as weekly or biweekly, or fractionated into smallerdoses and administered daily, semi-weekly, etc. to maintain an effectivedosage level.

[0053] Generally, a daily dosage of active ingredient (antibody) can beabout 0.1 to 100 mg/kg of body weight. Dosage forms suitable forinternal administration generally contain from about 0.1 mg to 500 mgsof active ingredient per unit. The active ingredient may vary from 0.5to 95% by weight based on the total weight of the composition. In somecases it may be desirable to limit the period of treatment due toexcessive T cell proliferation. The limitations will be empiricallydetermined, depending on the response of the patient to therapy, thenumber of T cells in the patient, etc. The number of T cells may bemonitored in a patient by methods known in the art, including stainingwith T cell specific antibodies and flow cytometry. The formulation isadministered at a dose effective to increase the response of T cells toantigenic stimulation. The determination of the T cell response willvary with the condition that is being treated. Useful measures of T cellactivity are proliferation, the release of cytokines, including. IL-2,IFNγ, TNFα, etc; T cell expression of markers such as CD25 and CD69; andother measures of T cell activity as known in the art.

[0054] The present invention further provides a kit that will allow theartisan to prepare an immunotherapeutic regimen for eliciting an immuneresponse against a p53-expressing malignancy. An example of a kitcomprises rMVAp53, a CTLA-4 blocking agent and/or a CpG ODN, andinstructions for using these compounds to elicit an immune responseagainst a p53-expressing malignancy in a subject. The kit may furthercomprise one or more pharmaceutically acceptable carriers. Whenadministered, the compositions of the kit are administered inpharmaceutically acceptable preparations. The terms administration,administering, and introducing refer to providing the compositions ofthe invention as a medicament to an individual in need of treatment orprevention of a p53-expressing malignancy. This medicament, whichcontains compositions of the present invention as the principal oractive ingredients, can be administered in a wide variety of therapeuticdosage forms in the conventional vehicles for topical, oral, systemic,local, and parenteral administration. Thus, the kits of the inventionprovide compositions for parenteral administration that comprise asolution of the compositions dissolved or-suspended in an acceptablecarrier, preferably an aqueous carrier. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like,including sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, and many others. Actual methods for preparing compounds forparenteral administration will be known or apparent to those skilled inthe art and are described in more detail in, for example, Remington: TheScience and Practice of Pharmacy (“Remington's Pharmaceutical Sciences”)Gennaro AR ed. 20^(th) edition, 2000: Williams & Wilkins PA, USA, whichis incorporated herein by reference.

[0055] Such preparations may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, supplementary immune potentiating agents such asadjuvants and cytokines and optionally other therapeutic agents. All thepreparations of the invention are administered in effective amounts. Aneffective amount is that amount of a pharmaceutical preparation thatalone, or together with further doses, stimulates the desired response.In the case of treating cancer, the desired response is inhibiting theinitiation or progression of the cancer, or producing regression of thecancer. This may involve only slowing the progression of the diseasetemporarily, although more preferably, it involves halting theprogression of the disease permanently. These desired responses can bemonitored by routine methods or can be monitored according to diagnosticmethods of the invention discussed herein. It is believed that doses ofimmunogens ranging from 10⁴ IU/kilogram to 10¹¹ IU/kilogram, dependingupon the mode of administration, would be effective. The preferred rangeis believed to be between 10⁶ IU and 10⁹ IU per kilogram. The absoluteamount will depend upon a variety of factors, including the combinationselected for administration, whether the administration is in single ormultiple doses, and individual patient parameters including age,physical condition, size, weight, and the stage of the disease. Thesefactors are well known to those of ordinary skill in the art and can beaddressed with no more than routine experimentation.

[0056] The following examples are provided to better illustrate theclaimed invention and are not to be interpreted as limiting the scope ofthe invention. To the extent that specific materials are mentioned, itis merely for purposes of illustration and is not intended to limit theinvention. Unless otherwise specified, general cloning procedures, suchas those set forth in Sambrook et al., Molecular Cloning, Cold SpringHarbor Laboratory (2001), Ausubel et al. (Eds.) Current Protocols inMolecular Biology, John Wiley & Sons (1986, 2000) are used. One skilledin the art may develop equivalent means or reactants without theexercise of inventive capacity and without departing from the scope ofthe invention.

[0057] It will be understood that many variations can be made in theprocedures herein described while still remaining within the bounds ofthe present invention. Likewise, it is understood that, due to thedegeneracy of the genetic code, nucleic acid sequences with codonsequivalent to those disclosed will encode functionally equivalent oridentical proteins as disclosed herein. It is the intention of theinventors that such variations are included within the scope of theinvention.

EXAMPLES

[0058] Materials and Methods

Animals

[0059] Female 6-8 week old Balb/c, C57BL/6, B6.129S2-IL6^(tm1Kopf)(IL-6⁺, and IFN-γ knock out (IFNγ^(KO)) mice on the Balb/c backgroundwere obtained from The Jackson Laboratory (Bar Harbor, Me.). TLR9^(−/−)mice were a kind gift from Dr. Shizuo Akira (Osaka University, Osaka,Japan). Mice were maintained in a specific pathogen-free environment.All studies were approved by the Research Animal Care Committee of theCity of Hope National Medical Center, and performed under the AAALACguidelines.

Cell Lines

[0060] CV-1 (Kit 1965), TK⁻ (Berson 1996), and Baby Hamster Kidney cells(BHK-21) (Macpherson 1962) were purchased from American Type CultureCollection (ATCC) (Manassas, Va.), and grown in MEM supplemented withnon-essential amino acids, L-glutamine, and 10% FCS. 11A-1 (Selvanayagam1995) was a kind gift from Dr. R. L. Ullrich (University of TexasMedical Branch, Galveston, Tex.). Hek 293 cells and p53null 10.1 cellswere kind gifts from Dr. K. K. Wong and Dr. Susan Kane (City of HopeNational Medical Center, Duarte, Calif.). MC-38 (Tan 1976) was a kindgift from Dr. S. A. Rosenberg (National Cancer Institute, Bethesda, Md.Meth A sarcoma cells (Meth A) (DeLeo 1977) were a kind gift from Dr. L.J. Old (Memorial Sloan-Kettering Cancer Center, New York, N.Y.). Meth Awas passaged as an ascitic tumor. Cells were harvested, counted andwashed with PBS prior to use. The characteristics of the Meth A, 11A-1,and MC-38 tumor cell lines are summarized in the following table: P53mutation Cell line Tumor MHC Background position(s) Meth A FibrosarcomaH-2^(d) 132, 168, 234 11A-1 Mammary cell H-2^(d) 173 carcinoma MC-38Colon carcinoma H-2^(b) 242

Antibodies

[0061] Anti-CD4 (GK1.5) (Dialynas 1983) and anti-NK1.1 (PK136) (Koo1984) were purchased from ATCC. Anti-CD8 (H35) (Miconnet 2001) andanti-CTLA-4 mAb (9H10) (Krummel 1995) were kind gifts from James P.Allison (University of California, Berkeley, Calif.). Antibodies wereproduced using a CELLine Device (BD Biosciences, Bedford, Mass.). IgGantibodies were purified by absorbance over protein G-Sepharose(Amersham, Uppsala, Sweden) followed by elution with 0.1 M Glycine-HCl,pH 2.7. The product was then dialyzed against phosphate-buffered normalsaline (PBS) and concentrated using a Centriplus centrifugal filterdevice (Millipore, Bedford, Mass.). Control Syrian Hamster IgG wasobtained from Jackson Immuno Research (West Grove, Pa.).

Viral Constructs

[0062] rMVA Expressing Murine p53 (rMVAmup53):

[0063] Wild type MVA (wtMVA) was obtained from Dr. Bernard Moss and Dr.Linda Wyatt (National Institutes of Health Bethesda, Md.). wtMVA stocksfor the generation of recombinant MVA (rMVA) containing mup53 arepropagated on specific pathogen free chicken embryo fibroblasts(SPF/CEF). The wtMVA stock is titrated by immunostaining, aliquoted, andstored at −80° C.

[0064] Murine p53 (mup53) is analogous to human p53, with 80% sequencehomology (Halevy 1991; Sukumar 1995). The mRNA coding sequence forfull-length wild type mup53 is shown in SEQ ID NO: 1. The level ofhomology between murine and human p53 makes the murine system anexcellent preclinical model for evaluating immunologic approaches forovercoming tolerance to p53. rMVA expressing murine p53 was generated byhomologous recombination of wtMVA and a pMCO3 insertion vectorcontaining a murine p53 insert, as described in Espenschied 2003. Theentire cDNA of murine wild type p53 was amplified by PCR of mRNAobtained from murine splenocytes. The murine p53 PCR product was ligatedinto the cloning site of the MVA expression vector pMCO3 (also obtainedfrom Dr. Moss and Dr. Wyatt). This vector contains sequences that insertinto deletion III of the MVA genome, and also contains the gus (E. coliB-glucuronidase) operon for screening purposes (Ourmanov 2000).Generation of recombinant MVA was achieved on monolayers of BHK-21 cells(Espenschied 2003). Briefly, BHK-21 cells were transfected with 20 μg ofplasmid DNA using Lipofectin (Invitrogen, Carlsbad, Calif.) and infectedwith wtMVA at an moi of 0.01. The infected cells were incubated for 48hours, then harvested, pelleted, and subjected to 3 cycles offreeze/thaw and sonication to lyse the cells. rMVA virus expressingmurine p53 (rMVAmup53) was screened for gus expression by adding X-GlcA(5-Bromo-4-Chloro-3-Indolyl B-D-Glucuronide, Sigma-Aldrich, St Louis,Mo.). After 10 rounds of purification, the rMVAmup53 was expanded onBHK-21 monolayers. The rMVAmup53 titer was determined by immunostaininginfected cultures using the Vectastain Elite ABC Kit (VectorLaboratories, Burlingame, Calif.).

[0065] rMVA Expressing Human p53 (rMVAhup53):

[0066] Two different constructs of rMVA expressing human p53 (rMVAhup53)were made. The mRNA sequence encoding full-length wild type hup53 isshown in SEQ ID NO: 2. The first was made using the pLW51 insertionplasmid, while the second was made using the pLW22 insertion plasmid.wtMVA used to make the first construct was propagated on SPF/CEF. wtMVAused to make the second construct was propagated on BHK-21 (BHK) cells.wtMVA stock was titrated by immunostaining, aliquoted, and stored at−80° C.

[0067] pLW51 was used as the insertion plasmid for generating the firstrMVAhup53 construct. pLW51 has four important features. First, itcontains MVA flanking regions of deletion III that allow it to insertinto the deletion III region of MVA via homologous recombination.Second, it contains a color screening marker gene, β-glucoronidase(gus), under control of a vaccinia promoter called P₁₁. Third, itcontains two direct repeats composed of MVA sequence (designated as DR1and DR2) flanking the gus screening marker gene to allow the gus gene tobe removed from recombinant MVA. Finally, it contains two vacciniapromoters (P_(SYN) and P_(7.5)) and two multiple cloning sites (MCS),permitting the insertion of two separate foreign genes under the controlof the P_(SYN) and P_(7.5) promoters. The first MCS is behind anearly/late PSYN promoter, while the second MCS uses an early/late PmH5promoter. This enables the elimination of the gus marker gene throughrecombination via a set of direct repeats, which flank it. Thegeneration of the initial rMVA stock is done on CEF utilizing methodsthat were previously described for BHK cells, with modifications toaccount for good laboratory practice (GLP) conditions. About 40-50 fociare pulled from the first rounds of screening to ensure that a correctrecombinant will be found, after which 5-10 are pulled in eachsubsequent round. After each round of selection, either immunostainingor immunofluorescence is performed on each plug to make sure that theplug is expressing the hup53 gene. To achieve a virus that will bedeleted of the bacterial gene marker, purified rMVA expressing hup53 isplated at low dilution in 24 well plates. Wells that do not have a colorreaction demonstrating the gus gene are further analyzed for thepresence of the hup53 gene product. This is accomplished by antibodystaining using conditions that allow recovery of the virus from thecells. Those wells that exhibit hup53 immunostaining in the absence of acolor reaction are further propagated and confirmed to be the correctphenotype. A portion of the viral plug pulled from the final round ofscreening absent the gus marker is expanded in a 100 mm tissue culturedish of CEF. This is followed by DNA extraction and PCR analysis(discussed below).

[0068] pLW22 was used as the insertion plasmid for generating the secondrMVAhup53 construct. pLW22 has MVA flanking regions that allow it toinsert into MVA via homologous recombination. It also has a colorscreening marker gene, β-galactosidase. To obtain DNA encoding wt hup53,pHp53B plasmid in E. coli was obtained from the ATCC (#57254). Hup53 wasamplified from the pHp53B plasmid using the forward primer of SEQ ID NO:3 and the reverse primer of SEQ ID NO: 4., Amplified wt hup53 DNA wasinserted into the pLW22 vector between restriction sites Pme-1 andAsc-1, generating pLW22-hup53. The plasmid sequence of pLW22-hup53 isshown in SEQ ID NO: 5.

[0069] Generation of rMVA was achieved on monolayers of BHK cells. BHKcells were transfected with 20 μg of plasmid DNA using Lipofectin(Invitrogen, Carlsbad, Calif.), and infected with wtMVA at an moi of0.01. The infected cells were incubated for 48 hours, then harvested,pelleted, and subjected to three cycles of freeze/thaw and sonication tolyse the cells. rMVA expressing hup53 was screened for β-gal expressionby adding presence of Bluo-gal™ substrate (Sigma-Aldrich, St Louis, Mo.)(Chakrabarti 1985). After 10 rounds of purification, the rMVAhup53 wasexpanded on BHK monolayers. The rMVA titer was determined byimmunostaining infected cultures using the Vectastain Elite ABC kit(Vector Laboratories, Burlingame, Calif.).

[0070] For both constructs, a standard DNA extraction is performed.Ethanol precipitation of 50 μL of the cell lysate resulted in enough DNAto run a PCR reaction to assure the absence of contaminating wtMVA. Oneset of PCR primers are designed outside the flanking regions of therecombination site for which the gene has been inserted. The presence ofunmodified wtMVA sequence will generate a 500 bp PCR product, whereasthe insertion of the sequence containing hup53 has a much largerfragment (>6 kb), which is usually difficult to amplify under standardPCR conditions. A second set of PCR primers are designed to amplify asequence within the hup53 insert. The presence of the hup53 insert willgenerate a 300 bp PCR product. The PCR samples are run on a 1% agarosegel and analyzed to determine if additional screenings are necessary toremove any remaining wtMVA. Examples of purified MVA containing humanp53 have been shown to be absolutely homogenous (FIG. 1).

[0071] rMVA Expressing pD65 (rMVApp65):

[0072] rMVA expressing pp65 (rMVApp65), a CMV tegument protein, wasconstructed using techniques similar to those used to constructrMVAmup53 (Gibson).

[0073] rVV Expressing Murine P53 or DD65:

[0074] Recombinant Western Reserve strain Vaccinia Virus expressingmurine wild type p53 or pp65 (rVVp53, rVVpp65) was constructed usingpublished techniques (Diamond 1997).

[0075] rAd Expressing Murine p53:

[0076] Recombinant adenovirus expressing wild type murine p53(rAd-mup53) was constructed using the pAd Easy system (He 1998). BothpAd Track-CMV and pAd Easy-1 plasmids were kindly provided by Dr. BertVogelstein (Johns Hopkins Oncology Center, Baltimore, Md.). Wild typemurine p53 cDNA was cloned into the Bgl II and Xba I site of a pAdTrack-CMV shuttle vector containing green fluorescent protein (GFP) witha CMV promoter (p53-pAd Track-CMV). The p53-pAd Track-CMV wascotransformed into BJ5183 cells with the pAd Easy-1 plasmid to generatethe p53 recombinant adenoviral construct by homologous recombination.The presence of the p53 gene in the recombinants was confirmed by DNAsequencing. The p53 recombinant adenoviral construct was cleaved withPac I and transfected into HEK-293 cells. rAd-mup53 was harvested 5 daysafter transfection and p53 protein expression was confirmed by westernblot. The adenovirus was expanded on HEK-293 cells and purified bycesium chloride gradient. The purified virus was dialyzed in PBS,titered on HEK-293 cells, and stored at −80° C. in 20% glycerol.

Oligodeoxynucleotides (ODN)

[0077] Synthetic ODN 1826 with CpG motifs (SEQ ID NO: 6) and non-CpG 6DN1982 (SEQ ID NO: 7) (Moldoveanu 1998), were synthesized withnuclease-resistant phosphorothioate backbones by Trilink (San Diego,Calif.). The Na⁺ salts of the ODNs were resuspended at 5 mg ml⁻¹ in 10mM Tris (pH 7.0) 1 mM EDTA and stored as 50 μl aliquots at −20° C.before dilution in aqueous 0.9% sodium chloride solution prior toinjection.

EXAMPLE 1 Expression of Murine p53 Protein By rMVAmup53

[0078] Expression of murine p53 protein following infection withrMVAmup53 was analyzed to determine the fidelity and extent of itsexpression from recombinant virus. Lysates were prepared from BHK or HEK293 cells infected with rMVAmup53 and subjected to SDS-PAGE and Westernblotting. Standard Western Blotting techniques were performed using anECL Western Blot Kit (Amersham Pharmacia Biotech, England). The sampleswere incubated with a purified mouse anti-p53 monoclonal antibody, PAb122 (Gurney 1980), followed by incubation with a peroxidase labeled goatanti-mouse secondary antibody provided in the ECL Western Blot kit.Western blot analysis of BHK cells infected with rMVAmup53 demonstratesabundant p53 expression (FIG. 2). The remarkable level of expressionexhibited by rMVAmup53 compared to other viral and cellular formsdemonstrates its usefulness in vaccination-protocols. As shown in FIG.1, the volume on the rMVAmup53 lane is between 80-160 fold less thanwhat was applied to the gel in the other lanes, yet the intensity of theband is several fold higher. This demonstrates a very high level of p53expression by rMVAmup53. Meth A cells were used as a positive controland BHK cells infected with HCMV IE1 exon 4 rMVA were used as negativecontrols. Meth A is a Balb/c derived, tumorigenic3-methylcholanthrene-induced sarcoma that over-expresses mutated p53. A53 kilodalton band was observed in both the p53 overexpressing Meth Asarcoma and the rMVAmup53 infected BHK cells (FIG. 1). This contrastswith the absence of 1-5 detectable p53 expression in the HCMV IE1 exon4-rMVA infected BHK cells. Strong p53 expression was also observed byfluorescence microscopy in BHK cells infected with rMVAmup53 (data notshown).

EXAMPLE 2 In vitro Generation of a p53-Specific CTL Response ByrMVAmup53

[0079] Vaccination of mice with rMVA expressing viral and tumorassociated antigens results in enhanced antigen specific CTL responses.One goal of this example was to determine if vaccination with rMVAmup53could break p53 tolerance, resulting in the generation of p53-specificCTL. Mice were vaccinated i.p. with 5×10⁷ pfu of either rMVAmup53 orrMVApp65. After two weeks, spleens were harvested and disassociated, andsplenocytes were washed and counted.

[0080] Splenocytes were restimulated in vitro for 6 days with syngeneicLPS blasts infected with rAd-mup53 or rMVAmup53. Na-⁵¹CrO₄-labeledtarget cells that overexpress wt p53 were added to 96 well plates withthe effectors, in triplicate, at various effector to target ratios, in200 μl of complete medium. The plates were incubated for 4 hours at 37°C., and the supernatant was harvested and analyzed. Percent specificlysis was calculated using the formula: percent specificrelease=(experimental release —spontaneous release)/(totalrelease−spontaneous release)×100. Splenocytes vaccinated with rMVAmup53recognized and lysed target cells that overexpressed wt p53 (FIG. 3). Incontrast, splenocytes from mice vaccinated with rMVApp65, whichstimulates a vigorous pp65 specific CTL response, did not recognize thep53 over-expressing targets (FIG. 3B), demonstrating the specificity ofthe lymphocyte response. rMVAmup53 vaccination can also stimulate CTLrecognition of a cell line bearing mutated p53, Meth A. Restimulatedsplenocytes vaccinated with rMVAmup53 recognized mutant p53over-expressing Meth A cells, but splenocytes vaccinated with rMVAmup53did not (FIG. 3c).

EXAMPLE 3 In Vivo rMVAmup53 Tumor Challenge Experiments

[0081] Since a single vaccination with rMVAmup53 resulted in enhancedCTL responses, there was sufficient justification to examine the effectof rMVAmup53 vaccination on the growth of tumor cells in vivo.

Statistical Methods

[0082] For experiments where the growth rate of some tumors necessitatedearly sacrifice, growth curves were compared by the time to a fixed sizeusing a logrank test. Contrasts of single groups to all others wereconducted after a single omnibus test. For cell depletion experiments,all mice were followed for a fixed amount of time, and final tumor sizewas compared by the Wilcoxon rank-sum test, after a significantKruskal-Wallis test if there were more than two groups. For survivalexperiments, a logrank test was used.

rMVAmuD53 vs. Meth A Cells

[0083] Six-week-old female Balb/c mice were injected by subcutaneous(s.c.) route in the left flank with 5×10⁵ Meth A cells. Mice injecteds.c. with Meth A cells develop a rapidly growing fibrosarcoma that killsthe majority of mice within 21 days (FIG. 3). On day 3, the mice werevaccinated with 5×10⁷ pfu of rMVAmup53 by intraperitoneal (i.p.)injection. Negative control mice were injected with 5×10⁷ rMVApp65 orPBS. The s.c. tumors were measured twice weekly in three dimensions withcalipers. Tumors in rMVAmup53 treated animals grew at a much slower ratethan those in control animals. At 14 days, the mean s.c. tumor volumefor the rMVAmup53 treated group (n=16) was dramatically lower than boththe rMVApp65 (n=16) and PBS (n=112) controls (22 mm³ versus 348 mm³,p<0.001 and 22 mm³ versus 252 mm³, p<0.001 by Student's t-test).Survival of rMVAmup53 treated animals was also significantly prolongedcompared to either control group (FIG. 4). 12 of the 16 rMVAmup53immunized mice failed to develop tumors entirely. The 12 tumor freerMVAmup53 treated animals were re-challenged at day 52 with 5×10⁵ Meth Atumor cells. All animals remained tumor free for the duration of a 30day observation period (data not shown).

rMVAmup53 Plus Anti-CTLA-4 mAb vs. Meth A Cells

[0084] One potent strategy for optimizing tumor vaccines involvesmanipulating negative regulation of T cell responsiveness using anantibody that blocks CTLA-4 engagement with ligand. This phenomenon hasbeen referred to as CTLA-4 blockade. Application of anti-CTLA-4 mAb incombination with cancer vaccines expressing tumor associatedautoantigens, in some cases, results in tumor rejection along withbreaking of tolerance and induction of autoimmunity. Therefore, mAbspecific to CTLA-4 was added to rMVAmup53 vaccination to determinewhether it would synergize and augment the anti-tumor activity againstMeth A in vivo. A more rigorous tumor model was designed in order toovercome the potent antitumor effect of CTLA-4 blockade alone.Six-week-old Balb/c mice were injected s.c. in the left flank with 10⁶Meth A cells rather than 5×10⁵ Meth A cells, and treatment was postponeduntil a palpable tumor nodule was identified (Day 6). This more rigorousmodel overcame the effect of the CTLA-4 blockade, producing a rapidlylethal tumor in the majority of mice despite anti-CTLA-4 mAb treatment(FIG. 5). On day 7, mice were injected i.p. with 5×10⁷ pfu of rMVAmup53.Controls were the same as above. Anti-CTLA-4 mAb antibody or controlhamster Ab were injected i.p. on days 6, 9, and 12 at 100, 50 and 50 μgdose, respectively. 11 of the 14 mice immunized with rMVAmup53 plusanti-CTLA-4 mAb rejected tumors, resulting in tumor free survival forthe duration of the 60 day observation period (FIG. 5). By contrast,mice treated with rMVApp65 and control antibody died rapidly ofprogressive tumor (FIG. 5) as did PBS treated controls (data not shown).The 11 tumor-free rMVAmup53 plus anti-CTLA-4 mAb treated mice alsorejected a re-challenge with 10⁶ Meth A tumor cells at 60 days, andremained tumor free for the duration of a 30 day observation period(data not shown).

rMVAmuD53 Plus Anti-CTLA-4 mAb vs. 11A-1-Cells

[0085] Six-week-old Balb/c mice were injected s.c. in the left flankwith 2×10⁶ 11A-1 cells. 11A-1 is a rapidly growing malignant cell linethat is poorly immunogenic. Mice vaccinated with 10⁶ irradiated 11A-1tumor cells failed to reject a subsequent challenge with 11A-1 (data notshown). Anti-CTLA4 mAb or the control hamster antibody was injected i.p.on days 4, 7, and 10 at 100, 50, and 50 μg/dose, respectively. On day 5,mice were vaccinated i.p. with either 5×10⁷ pfu of rMVAmup53, 5×10⁷MVApp65, or PBS. s.c. tumors were measured twice weekly in threedimensions with calipers. Mice vaccinated with rMVAmup53 plusanti-CTLA-4 mAb rejected their tumors (FIG. 6). Animals treated withanti-CTLA4 mAb alone or with a control MVA vaccine developed rapidlyprogressing lethal tumors (p 0.00044, comparing rMVAmup53 withanti-CTLA-4 mAb blockade to control groups).

rMVAmup53 Plus Anti-CTLA-4 mAb vs. MC-38 Cells

[0086] Six-week-old C57BL/6 mice, TLR9^(−/−), or IL-6^(−/−) mice wereinjected s.c. in the left flank with 1×10⁶ MC-38 cells. Anti-CTLA-4 mAbor the control hamster antibody was injected i.p. on days 4, 7, and 10at 100, 50, and 50 μg/dose, respectively. On day 5, mice were vaccinatedi.p. with either 5×10⁷ pfu of rMVAmup53, 5×10⁷ rMVApp65, or PBS. s.c.tumors were measured twice weekly in three dimensions with calipers.Mice vaccinated with rMVAmup53 plus anti-CTLA-4 mAb rejected theirtumors, while those treated with anti-CTLA-4 mAb alone or with a controlMVA vaccine developed rapidly progressing tumors (p=0.0001, comparingrMVAmup53 with anti-CTLA-4 mAb to control groups) (FIG. 7).

rMVAmup53 Plus CpG ODN vs. 11A-1 Cells

[0087] CpG ODN treatment has been shown to be an effectiveimmunomodulator in a number of experimental tumor vaccine models (Krieg2002). Mice were challenged with 11A-1 tumor as above. 15 nmoles of CpGODN or the non-CpG ODN control were injected i.p. on days 4, 9, and 14.On day 5, the mice were vaccinated i.p. with either 5×10⁷ pfu ofrMVAmup53, 5×10⁷ rMVApp65, or PBS. The s.c. tumors were measured twiceweekly in three dimensions with calipers. While rMVAmup53 and CpG ODNeach separately resulted in minimal attenuation of tumor growth, allanimals developed progressively lethal tumors. The combination of CpGODN and rMVAmup53 vaccination resulted in significantly diminished tumoroutgrowth (p=0.00002) (FIG. 8). 6 of the 8 animals treated withrMVAmup53 plus CpG ODN did not develop palpable tumors and developedlasting tumor immunity, rejecting a rechallenge with 11A-1 at 60 days(data not shown).

rMVAmup53 Plus CpG ODN vs. Meth A Cells

[0088] A pattern of tumor rejection similar to that for 11A-1 was seenfollowing treatment of early established Meth A tumors in Balb/c mice(p=0.0015) (FIG. 9).

rMVAmuD53 Plus CpG ODN vs. MC-38 Cells

[0089] To demonstrate that the immunomodulator effect of CpG ODN onrMVAmup53 vaccination is not strain specific, the vaccination strategywas repeated in C57BL/6 mice bearing early established MC38 coloncancers. Vaccination with rMVAmup53 plus CpG ODN resulted in significantsuppression of tumor growth (p=0.0004) (FIG. 10).

rMVAmup53 Plus Anti-CTLA-4 mAb Plus CpG ODN vs. 11A-1 Cells

[0090] A more rigorous tumor model was designed to evaluate thepotential additive effects of CpG ODN and anti-CTLA-4 mAb on rMVAmup53vaccination. Six-week-old Balb/c mice were injected s.c. in the leftflank with 2×10⁶11A-1 cells and followed for two weeks until palpabletumors were present. Anti-CTLA4 mAb or the control hamster antibody wasinjected i.p. on days 14, 17, and 20, at 100, 50, and 50 μg/dose,respectively. 15 nmoles of CpG ODN was injected i.p. on days 14, 19, and24. On day 15, the mice were vaccinated i.p. with either 5×10⁷ pfu ofrMVAmup53, 5×10⁷ MVApp65, or PBS.

[0091] rMVAmup53 vaccination combined with either anti-CTLA-4 mAb or CpGODN immunomodulators resulted in prolonged survival, but all animalseventually succumbed to progressive tumor growth. The combination ofanti-CTLA-4 mAb and CpG ODN administration with rMVAmup53 vaccinationresulted in tumor rejection and prolonged survival in the majority oftreated animals (FIG. 11). The combination of anti-CTLA-4 mAb and CpGODN provides better immunomodulator activity than either CpG ODN alone(p=0.02) or anti-CTLA-4 mAb alone (p=0.01). The effect of combinedanti-CTLA4 mAb and CpG ODN administration provides a greater benefit interms of survival at 60 days than the simple addition of the effects ofboth immunomodulators separately.

rMVAmup53 Plus Anti-CTLA-4 mAb Plus CpG ODN vs. MC-38 Cells

[0092] A similar pattern was seen in C57BL/6 mice bearing MC 38 tumors(FIG. 12). C57BL/6 mice bearing MC-38 tumors were treated with rMVAmup53plus a combination of anti-CTLA-4 mAb and CpG ODN as described above for11A-1. In this tumor model, the combination of anti-CTLA-4 mAb and CpGODN also provided better immunomodulator activity than either CpG ODNalone (p=0.002) or anti-CTLA-4 mAb alone (p=0.001). The combined effectin both tumor models is not simply a dose additive effect, as the CpGODN and anti-CTLA-4 mAb were both already administered at doses ofmaximal efficacy. The striking increases in activity found when bothimmunomodulators are used together in at least two different tumorssuggests that further investigation of the combined modality iswarranted in humans.

EXAMPLE 4 Cellular Requirements for Anti-CTLA-4 mAb and CpG ODNImmunomodulator Effect

[0093] To determine the cellular requirements for the immunomodulatoreffect of anti-CTLA-4 mAb and CpG ODN, Balb/c mice were depleted ofCD4⁺, CD8⁺, or NK cells prior to vaccination. Depletion was accomplishedby i.p. injection of 200 μg of CD4⁺, CD8⁺, or NK1.1 cell specific mAbs,or a control mAb. Injections were given on days −1, 1, 3, 4, 6, 8, and15, with a maintenance dose every 7 days until the termination of theanimals. This regimen was shown to deplete (>95%) Balb/c mice ofCD4^(+, CD)8⁺, or NK 1.1 cells based on flow cytometry of peripheralblood from treated animals (data not shown).

[0094] The cellular requirements for the immunomodulator effect of CTLA4blockade on rMVAmup53 vaccination were evaluated using the Meth A tumormodel in Balb/c mice. Mice depleted of CD8⁺ T cells or CD4^(+ and CD)8⁺T cells simultaneously develop rapidly lethal tumors. These tumors areresistant to vaccination with rMVAmup53 and anti-CTLA4 mAb. In contrast,CD4⁺ T cell depletion resulted in only a partial abrogation of responseto the vaccine. NK1.1 cell depletion had little effect on the ability ofvaccinated mice to reject Meth A (FIG. 13a). Results were the same whenthe depleting mAbs were administered after vaccine and anti-CTLA-4 mAbtreatment (data now shown). Similar results were also obtained when the11A-1 tumor model was used rather than the Meth A tumor model. Thetherapeutic effect of rMVAmup53, and anti-CTLA-4 mAb could be eliminatedby administering depleting doses of anti-CD8⁺ mAb (p=0.004) (FIG. 15).The antitumor effect was partially blocked by the administration ofdepleting anti-CD4⁺ mAb (p=0.008), and unaffected by the administrationof an NK depleting mAb. These results show that the immunomodulatoreffect of anti-CTLA-4 mAb is entirely dependent on CD8⁺ cells, partiallydependent on CD4⁺ cells, and not dependent at all on NK cells(Espenschied 2003).

[0095] The cellular requirements for the immunomodulator effect of CpGODN on rMVAmup53 vaccination were evaluated using Balb/c mice withfour-day established 11A-1 tumors. As with anti-CTLA-4 mAb, theimmunomodulator effect of CpG ODN on MVAmup53 vaccination could becompletely abrogated by the administration of depleting CD8⁺ mAb(p=0.004) (FIG. 14). However, unlike anti-CTLA-4 mAb, theimmunomodulator effect of CpG ODN was unaffected by CD4⁺ depletion,while depletion of NK cells partially abrogated the vaccine effect(p=0.007, comparing NK to CD4⁺ and control antibody depletions). Thedifference in cellular requirements for CD4⁺ and NK between anti-CTLA-4mAb and CpG ODN is striking, because both immunomodulators causeequivalent levels of rejection. These results suggest that the twoimmunomodulators act through differing immunologic mechanisms. Thisinformation, combined with the data regarding the effects of combinedanti-CTLA-4 mAb/CpG ODN administration on rMVAmup53, suggest asynergistic effect by the two immunomodulators on tumor growth.

Contribution of IFN-γ

[0096] The contribution of IFN-γ secretion to the effect of CTLA4blockade and rMVAmup53 vaccination was evaluated in IFNγ^(KO) mice. Bothunvaccinated mice and mice vaccinated with rMVApp65 and anti-CTLA-4 mAbdeveloped lethal tumors at a rate similar to that seen in normal Balb/cmice (FIG. 13b). 3 of the 5 IFNγ^(KO) mice that were vaccinated withrMVAmup53 and anti-CTLA-4 mAb developed lethal tumor growth, confirminga contribution of IFN-γ to the vaccine/CTLA4 blockade effect.

Contribution of TLR 9

[0097] The cell subset depletion studies suggest that the mechanism ofimmunomodulator activity of CTLA-4 blockade and CpG ODN is different.CpG ODN activity results from the stimulation of B-cells andplasmacytoid dendritic cells through an interaction with the TLR9receptor (Chu 1997). CpG treatment causes a bias towards the TH1cytokine milieu and stimulation of NK cell proliferation, which mayaccount for the partial effect on tumor rejection. To further delineatethe divergent pathways involved in the CpG ODN and CTLA-4 blockadeimmunomodulator effects, MC-38 tumor challenge experiments wereconducted in TLR9^(−/−) mice. TLR9^(−/−) mice fail to immunologicallyrespond to CpG ODN administration (Hemmi 2000). As expected, TLR9^(−/−)mice bearing early established MC-38 tumors failed to immunologicallyrespond to CpG ODN and rMVAmup53 vaccination (FIG. 16). In contrast,inclusion of anti-CTLA-4 mAb with rMVAmup53 vaccination resulted intumor rejection in TLR9^(−/−) mice (p=0.0009) that was similar to thatseen in wt C57BL/6 mice (FIG. 16, FIG. 7).

Contribution of IL-6

[0098] Both CpG ODN and CTLA-4 blockade inhibit CD25⁺ CD4⁺ suppressor orregulatory T cells (Treg), and this effect may contribute to theirimmunomodulator activity in the described tumor models. Blocking CTLA-4is thought to have a direct inhibitory affect on Tregs, most of whichconstitutively express CTLA-4 (Read 2000). In contrast, CpG ODN inhibitsTreg activity through the secretion of IL-6 by DC (Pasare 2003). Toevaluate the role of IL-6 on the CpG ODN and anti-CTLA-4 mAbimmunomodulator effects, tumor challenge experiments were conducted inIL-6^(−/−) mice. IL-6^(−/−) mice bearing early established MC-38 tumorsfailed to immunologically respond to rMVAmup53 vaccination with CpG ODNby rejecting tumor (FIG. 17). This suggests that CpG ODN could bemediating its immunomodulator effects, at least in part, through theIL-6 dependent pathway of Treg cell inhibition. In contrast, anti-CTLA-4mAB inclusion with rMVAmup53 vaccination resulted in tumor rejection inIL-6^(−/−) mice (p=0.02) to an extent similar to that seen in wt C57BL/6mice (FIG. 17, FIG. 7).

EXAMPLE 5 Expression of Human p53 By rMVAhup53

[0099] BHK cells were infected with purified rMVAhup53. Expression ofhup53 was measured at 24 and 48 hours, and analyzed by Western blot andimmunohistochemistry. The infected rMVAhup53 cells demonstrated vigorousexpression of hup53 at both time periods (FIG. 18).

EXAMPLE 6 In vivo rMVAhu53 Tumor Challenge Experiments

[0100] Hupki mice, a novel murine knock-in model expressing human p53,were obtained from Dr. Monica Hollstein (DKFZ, Heidelberg, Germany) inthe 129/Sv genetic background. The mice were backcrossed for 4generations onto the Balb/c(H-2^(d)) background in order to takeadvantage of the knock-in transgene in a murine background where tumorsand other reagents are readily available. The hupki mice on the Balb/cbackground were backcrossed to homozygosity as confirmed by PCRanalysis, using a mating procedure that minimized inbreeding effects(data not shown). The 4T1 (H-2^(d)) murine breast carcinoma cell linewas stably transfected with human p53, and hupki mice were s.c. injectedwith 5×10⁴ 4T1/hup53 in the flank. Mice injected with 4T1/hup53 growprogressive tumors, and the majority succumb to these tumors by day 35.To test the efficacy of rMVAhup53, mice were vaccinated with 10⁷ pfurMVAhup53 by i.p. injection on day 6 after 4T1/hup53 injection. Ten dayslater, the mice received an rMVAhup53 booster injection, along withCpG-ODN (15 nmole of ODN 1826) and anti-CTLA-4 mAb (50 μg/mouse).rMVAhup53 vaccination resulted in a statistically significantimprovement in survival (p<0.05, two sided T-test) compared to PBScontrols (FIG. 19).

[0101] As stated above, the foregoing are merely intended to illustratethe various embodiments of the present invention. As such, the specificmodifications discussed above are not to be construed as limitations onthe scope of the invention. It will be apparent to one skilled in theart that various equivalents, changes, and modifications may be madewithout departing from the scope of the invention, and it is understoodthat such equivalent embodiments are to be included herein. Allreferences cited herein are incorporated by reference as if fully setforth herein.

[0102] Abbreviations used herein: GFP, green fluorescent protein; DC,dendritic cells; IFN-γ^(KO), IFN-γ knock out; MVA, modified vacciniavirus Ankara; rMVA, recombinant modified vaccinia virus Ankara;rAd-mup53, recombinant Adenovirus expressing murine wild type p53;hup53, wild type human p53; mup53, wild type murine p53; rMVAp53,recombinant MVA expressing p53; rMVAmup53, recombinant MVA expressingwild type murine p53; rMVAhup53, recombinant MVA expressing wild typehuman p53; rMVApp65, recombinant MVA expressing pp65; rVVmup53,recombinant vaccinia virus expressing murine wild type p53; rWpp65,recombinant vaccinia virus expressing pp65; wtMVA, wild type MVA; WR,Western Reserve; i.p., intraperitoneal; s.c., subcutaneous; mAb,monoclonal antibody.

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1 7 1 1173 DNA Mus musculus 1 atgactgcca tggaggagtc acagtcggatatcagcctcg agctccctct gagccaggag 60 acattttcag gcttatggaa actacttcctccagaagata tcctgccatc acctcactgc 120 atggacgatc tgttgctgcc ccaggatgttgaggagtttt ttgaaggccc aagtgaagcc 180 ctccgagtgt caggagctcc tgcagcacaggaccctgtca ccgagacccc tgggccagtg 240 gcccctgccc cagccactcc atggcccctgtcatcttttg tcccttctca aaaaacttac 300 cagggcaact atggcttcca cctgggcttcctgcagtctg ggacagccaa gtctgttatg 360 tgcacgtact ctcctcccct caataagctattctgccagc tggtgaagac gtgccctgtg 420 cagttgtggg tcagcgccac acctccagctgggagccgtg tccgcgccat ggccatctac 480 aagaagtcac agcacatgac ggaggtcgtgagacgctgcc cccaccatga gcgctgctcc 540 gatggtgatg gcctggctcc tccccagcatcttatccggg tggaaggaaa tttgtatccc 600 gagtatctgg aagacaggca gacttttcgccacagcgtgg tggtacctta tgagccaccc 660 gaggccggct ctgagtatac caccatccactacaagtaca tgtgtaatag ctcctgcatg 720 gggggcatga accgccgacc tatccttaccatcatcacac tggaagactc cagtgggaac 780 cttctgggac gggacagctt tgaggttcgtgtttgtgcct gccctgggag agaccgccgt 840 acagaagaag aaaatttccg caaaaaggaagtcctttgcc ctgaactgcc cccagggagc 900 gcaaagagag cgctgcccac ctgcacaagcgcctctcccc cgcaaaagaa aaaaccactt 960 gatggagagt atttcaccct caagatccgcgggcgtaaac gcttcgagat gttccgggag 1020 ctgaatgagg ccttagagtt aaaggatgcccatgctacag aggagtctgg agacagcagg 1080 gctcactcca gctacctgaa gaccaagaagggccagtcta cttcccgcca taaaaaaaca 1140 atggtcaaga aagtggggcc tgactcagactga 1173 2 1182 DNA Homo sapiens 2 atggaggagc cgcagtcaga tcctagcgtcgagccccctc tgagtcagga aacattttca 60 gacctatgga aactacttcc tgaaaacaacgttctgtccc ccttgccgtc ccaagcaatg 120 gatgatttga tgctgtcccc ggacgatattgaacaatggt tcactgaaga cccaggtcca 180 gatgaagctc ccagaatgcc agaggctgctccccgcgtgg cccctgcacc agcagctcct 240 acaccggcgg cccctgcacc agccccctcctggcccctgt catcttctgt cccttcccag 300 aaaacctacc agggcagcta cggtttccgtctgggcttct tgcattctgg gacagccaag 360 tctgtgactt gcacgtactc ccctgccctcaacaagatgt tttgccaact ggccaagacc 420 tgccctgtgc agctgtgggt tgattccacacccccgcccg gcacccgcgt ccgcgccatg 480 gccatctaca agcagtcaca gcacatgacggaggttgtga ggcgctgccc ccaccatgag 540 cgctgctcag atagcgatgg tctggcccctcctcagcatc ttatccgagt ggaaggaaat 600 ttgcgtgtgg agtatttgga tgacagaaacacttttcgac atagtgtggt ggtgccctat 660 gagccgcctg aggttggctc tgactgtaccaccatccact acaactacat gtgtaacagt 720 tcctgcatgg gcggcatgaa ccggaggcccatcctcacca tcatcacact ggaagactcc 780 agtggtaatc tactgggacg gaacagctttgaggtgcatg tttgtgcctg tcctgggaga 840 gaccggcgca cagaggaaga gaatctccgcaagaaagggg agcctcacca cgagctgccc 900 ccagggagca ctaagcgagc actgtccaacaacaccagct cctctcccca gccaaagaag 960 aaaccactgg atggagaata tttcacccttcagatccgtg ggcgtgagcg cttcgagatg 1020 ttccgagagc tgaatgaggc cttggaactcaaggatgccc aggctgggaa ggagccaggg 1080 gggagcaggg ctcactccag ccacctgaagtccaaaaagg gtcagtctac ctcccgccat 1140 aaaaaactca tgttcaagac agaagggcctgactcagact ga 1182 3 41 DNA Artificial forward primer for amplificationof wt human p53 3 agctttgttt aaacgccacc acccacgctt ccctggattg g 41 4 37DNA artificial reverse primer for amplification of wt human p53 4ttggcgcgcc tttatttcag tctgagtcag gcccttc 37 5 8618 DNA Artificial pLW22plasmid containing wt human p53 insert 5 cctcctgaaa aactggaatttaatacacca tttgtgttca tcatcagaca tgatattact 60 ggatttatat tgtttatgggtaaggtagaa tctccttaat atgggtacgg tgtaaggaat 120 cattatttta tttatattgatgggtacgtg aaatctgaat tttcttaata aatattattt 180 ttattaaatg tgtatatgttgttttgcgat agccatgtat ctactaatca gatctattag 240 agatattatt aattctggtgcaatatgaca aaaattatac actaattagc gtctcgtttc 300 agacatggat ctgtcacgaattaatacttg gaagtctaag cagctgaaaa gctttctctc 360 tagcaaagat gcatttaaggcggatgtcca tggacatagt gccttgtatt atgcaatagc 420 tgataataac gtgcgtctagtatgtacgtt gttgaacgct ggagcattga aaaatcttct 480 agagaatgaa tttccattacatcaggcagc cacattggaa gataccaaaa tagtaaagat 540 tttggctatt cagtggactggatgattcga ggtacccgat cccccctgcc cggttattat 600 tatttttgac accagaccaactggtaatgg tagcgaccgg cgctcagctg aattccgccg 660 atactgacgg gctccaggagtcgtcgccac caatccccat atggaaaccg tcgatattca 720 gccatgtgcc ttcttccgcgtgcagcagat ggcgatggct ggtttccatc agttgctgtt 780 gactgtagcg gctgatgttgaactggaagt cgccgcgcca ctggtgtggg ccataattca 840 attcgcgcgt cccgcagcgcagaccgtttt cgctcgggaa gacgtacggg gtatacatgt 900 ctgacaatgg cagatcccagcggtcaaaac aggcggcagt aaggcggtcg ggatagtttt 960 cttgcggccc taatccgagccagtttaccc gctctgctac ctgcgccagc tggcagttca 1020 ggccaatccg cgccggatgcggtgtatcgc tcgccacttc aacatcaacg gtaatcgcca 1080 tttgaccact accatcaatccggtaggttt tccggctgat aaataaggtt ttcccctgat 1140 gctgccacgc gtgagcggtcgtaatcagca ccgcatcagc aagtgtatct gccgtgcact 1200 gcaacaacgc tgcttcggcctggtaatggc ccgccgcctt ccagcgttcg acccaggcgt 1260 tagggtcaat gcgggtcgcttcacttacgc caatgtcgtt atccagcggt gcacgggtga 1320 actgatcgcg cagcggcgtcagcagttgtt ttttatcgcc aatccacatc tgtgaaagaa 1380 agcctgactg gcggttaaattgccaacgct tattacccag ctcgatgcaa aaatccattt 1440 cgctggtggt cagatgcgggatggcgtggg acgcggcggg gagcgtcaca ctgaggtttt 1500 ccgccagacg ccactgctgccaggcgctga tgtgcccggc ttctgaccat gcggtcgcgt 1560 tcggttgcac tacgcgtactgtgagccaga gttgcccggc gctctccggc tgcggtagtt 1620 caggcagttc aatcaactgtttaccttgtg gagcgacatc cagaggcact tcaccgcttg 1680 ccagcggctt accatccagcgccaccatcc agtgcaggag ctcgttatcg ctatgacgga 1740 acaggtattc gctggtcacttcgatggttt gcccggataa acggaactgg aaaaactgct 1800 gctggtgttt tgcttccgtcagcgctggat gcggcgtgcg gtcggcaaag accagaccgt 1860 tcatacagaa ctggcgatcgttcggcgtat cgccaaaatc accgccgtaa gccgaccacg 1920 ggttgccgtt ttcatcatatttaatcagcg actgatccac ccagtcccag acgaagccgc 1980 cctgtaaacg gggatactgacgaaacgcct gccagtattt agcgaaaccg ccaagactgt 2040 tacccatcgc gtgggcgtattcgcaaagga tcagcgggcg cgtctctcca ggtagcgaaa 2100 gccatttttt gatggaccatttcggcacag ccgggaaggg ctggtcttca tccacgcgcg 2160 cgtacatcgg gcaaataatatcggtggccg tggtgtcggc tccgccgcct tcatactgca 2220 ccgggcggga aggatcgacagatttgatcc agcgatacag cgcgtcgtga ttagcgccgt 2280 ggcctgattc attccccagcgaccagatga tcacactcgg gtgattacga tcgcgctgca 2340 ccattcgcgt tacgcgttcgctcatcgccg gtagccagcg cggatcatcg gtcagacgat 2400 tcattggcac catgccgtgggtttcaatat tggcttcatc caccacatac aggccgtagc 2460 ggtcgcacag cgtgtaccacagcggatggt tcggataatg cgaacagcgc acggcgttaa 2520 agttgttctg cttcatcagcaggatatcct gcaccatcgt ctgctcatcc atgacctgac 2580 catgcagagg atgatgctcgtgacggttaa cgcctcgaat cagcaacggc ttgccgttca 2640 gcagcagcag accattttcaatccgcacct cgcggaaacc gacatcgcag gcttctgctt 2700 caatcagcgt gccgtcggcggtgtgcagtt caaccaccgc acgatagaga ttcgggattt 2760 cggcgctcca cagtttcgggttttcgacgt tgagacgtag tgtgacgcga tcggcataac 2820 caccacgctc atcgataatttcaccgccga aaggcgcggt gccgctggcg acctgcgttt 2880 caccctgcca taaagaaactgttacccgta ggtagtcacg caactcgccg cacatctgaa 2940 cttcagcctc cagtacagcgcggctgaaat catcattaaa gcgagtggca acatggaaat 3000 cgctgatttg tgtagtcggtttatgcagca acgagacgtc acggaaaatg ccgctcatcc 3060 gccacatatc ctgatcttccagataactgc cgtcactcca acgcagcacc atcaccgcga 3120 ggcggttttc tccggcgcgtaaaaatgcgc tcaggtcaaa ttcagacggc aaacgactgt 3180 cctggccgta accgacccagcgcccgttgc accacagatg aaacgccgag ttaacgccat 3240 caaaaataat tcgcgtctggccttcctgta gccagctttc atcaacatta aatgtgagcg 3300 agtaacaacc cgtcggattctccgtgggaa caaacggcgg attgaccgta atgggatagg 3360 ttacgttggt gtagatgggcgcatcgtaac cgtgcatctg ccagtttgag gggacgacga 3420 cagtatcggc ctcaggaagatcgcactcca gccagctttc cggcaccgct tctggtgccg 3480 gaaaccaggc aaagcgccattcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg 3540 tgcgggcctc ttcgctattacgccagctgg cgaaaggggg atgtgctgca aggcgattaa 3600 gttgggtaac gccagggttttcccagtcac gacgttgtaa aacgacggga tctcccatgc 3660 tcgagttatg atctacttccttaccgtgca ataaattaga atatattttc tacttttacg 3720 agaaattaat tattgtatttattatttatg ggtgaaaaac ttactataaa aagcgggtgg 3780 gtttggaatt agtgaaagctgggagatctg gcgcgccttt atttcagtct gagtcaggcc 3840 cttctgtctt gaacatgagttttttatggc gggaggtaga ctgacccttt ttggacttca 3900 ggtggctgga gtgagccctgctcccccctg gctccttccc agcctgggca tccttgagtt 3960 ccaaggcctc attcagctctcggaacatct cgaagcgctc acgcccacgg atctgaaggg 4020 tgaaatattc tccatccagtggtttcttct ttggctgggg agaggagctg gtgttgttgg 4080 gcagtgctcg cttagtgctccctgggggca gctcgtggtg aggctcccct ttcttgcgga 4140 gattctcttc ctctgtgcgccggtctctcc caggacaggc acaaacacgc acctcaaagc 4200 tgttccgtcc cagtagattaccactggagt cttccagtgt gatgatggtg aggatgggcc 4260 tccggttcat gccgcccatgcaggaactgt tacacatgta gttgtagtgg atggtggtac 4320 agtcagagcc aacctcaggcggctcatagg gcaccaccac actatgtcga aaagtgtttc 4380 tgtcatccaa atactccacacgcaaatttc cttccactcg gataagatgc tgaggagggg 4440 ccagaccatc gctatctgagcagcgctcat ggtgggggca gcgcctcaca acctccgtca 4500 tgtgctgtga ctgcttgtagatggccatgg cgcggacgcg ggtgccgggc gggggtgtgg 4560 aatcaaccca cagctgcacagggcaggtct tggccagttg gcaaaacatc ttgttgaggg 4620 caggggagta cgtgcaagtcacagacttgg ctgtcccaga atgcaagaag cccagacgga 4680 aaccgtagct gccctggtaggttttctggg aagggacaga agatgacagg ggccaggagg 4740 gggctggtgc aggggccgccggtgtaggag ctgctggtgc aggggccacg gggggagcag 4800 cctctggcat tctgggagcttcatctggac ctgggtcttc agtgaaccat tgttcaatat 4860 cgtccgggga cagcatcaaatcatccattg cttgggacgg caagggggac agaacgttgt 4920 tttcaggaag tagtttccataggtctgaaa atgtttcctg actcagaggg ggctcgacgc 4980 taggatctga ctgcggctcctccatggcag tgacccggaa ggcagtctgg ctgccaatcc 5040 agggaagcgt gggtggtggcgtttaaacgg atcccgagct tatttatatt ccaaaaaaaa 5100 aaaataaaat ttcaatttttaagctgggga tcctctagag tcgacctgca ggcatgctcg 5160 agcggccgcc agtgtgatggatatctgcag aattcggctt ggggggctgc aggtggatgc 5220 gatcatgacg tcctctgcaatggataacaa tgaacctaaa gtactagaaa tggtatatga 5280 tgctacaatt ttacccgaaggtagtagcat ggattgtata aacagacaca tcaatatgtg 5340 tatacaacgc acctatagttctagtataat tgccatattg gatagattcc taatgatgaa 5400 caaggatgaa ctaaataatacacagtgtca tataattaaa gaatttatga catacgaaca 5460 aatggcgatt gaccattatggagaatatgt aaacgctatt ctatatcaaa ttcgtaaaag 5520 acctaatcaa catcacaccattaatctgtt taaaaaaata aaaagaaccc ggtatgacac 5580 ttttaaagtg gatcccgtagaattcgtaaa aaaagttatc ggatttgtat ctatcttgaa 5640 caaatataaa ccggtttatagttacgtcct gtacgagaac gtcctgtacg atgagttcaa 5700 atgtttcatt gactacgtggaaactaagta tttctaaaat taatgatgca ttaatttttg 5760 tattgattct caatcctaaaaactaaaata tgaataagta ttaaacatag cggtgtacta 5820 attgatttaa cataaaaaatagttgttaac taatcatgag gactctactt attagatata 5880 ttctttggag aaatgacaacgatcaaaccg ggcatgcaag cttgtctccc tatagtgagt 5940 cgtattagag cttggcgtaatcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 6000 tcacaattcc acacaacatacgagccggaa gcataaagtg taaagcctgg ggtgcctaat 6060 gagtgagcta actcacattaattgcgttgc gctcactgcc cgctttcgag tcgggaaacc 6120 tgtcgtgcca gctgcattaatgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 6180 ggcgctcttc cgcttcctcgctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 6240 cggtatcagc tcactcaaaggcggtaatac ggttatccac agaatcaggg gataacgcag 6300 gaaagaacat gtgagcaaaaggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 6360 tggcgttttt cgataggctccgcccccctg acgagcatca caaaaatcga cgctcaagtc 6420 agaggtggcg aaacccgacaggactataaa gataccaggc gtttccccct ggaagctccc 6480 tcgtgcgctc tcctgttccgaccctgccgc ttaccggata cctgtccgcc tttctccctt 6540 cgggaagcgt ggcgctttctcatagctcac gctgtaggta tctcagttcg gtgtaggtcg 6600 ttcgctccaa gctgggctgtgtgcacgaac cccccgttca gcccgaccgc tgcgccttat 6660 ccggtaacta tcgtcttgagtccaacccgg taagacacga cttatcgcca ctggcagcag 6720 ccactggtaa caggattagcagagcgaggt atgtaggcgg tgctacagag ttcttgaagt 6780 ggtggcctaa ctacggctacactagaagga cagtatttgg tatctgcgct ctgctgaagc 6840 cagttacctt cggaaaaagagttggtagct cttgatccgg caaacaaacc accgctggta 6900 gcggtggttt ttttgtttgcaagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 6960 atcctttgat cttttctacggggtctgacg ctcagtggaa cgaaaactca cgttaaggga 7020 ttttggtcat gagattatcaaaaaggatct tcacctagat ccttttaaat taaaaatgaa 7080 gttttaaatc aatctaaagtatatatgagt aaacttggtc tgacagttac caatgcttaa 7140 tcagtgaggc acctatctcagcgatctgtc tatttcgttc atccatagtt gcctgactcc 7200 ccgtcgtgta gataactacgatacgggagg gcttaccatc tggccccagt gctgcaatga 7260 taccgcgaga cccacgctcaccggctccag atttatcagc aataaaccag ccagccggaa 7320 gggccgagcg cagaagtggtcctgcaactt tatccgcctc catccagtct attaattgtt 7380 gccgggaagc tagagtaagtagttcgccag ttaatagttt gcgcaacgtt gttggcattg 7440 ctacaggcat cgtggtgtcacgctcgtcgt ttggtatggc ttcattcagc tccggttccc 7500 aacgatcaag gcgagttacatgatccccca tgttgtgcaa aaaagcggtt agctccttcg 7560 gtcctccgat cgttgtcagaagtaagttgg ccgcagtgtt atcactcatg gttatggcag 7620 cactgcataa ttctcttactgtcatgccat ccgtaagatg cttttctgtg actggtgagt 7680 actcaaccaa gtcattctgagaatagtgta tgcggcgacc gagttgctct tgcccggcgt 7740 caatacggga taataccgcgccacatagca gaactttaaa agtgctcatc attggaaaac 7800 gttcttcggg gcgaaaactctcaaggatct taccgctgtt gagatccagt tcgatgtaac 7860 ccactcgtgc acccaactgatcttcagcat cttttacttt caccagcgtt tctgggtgag 7920 caaaaacagg aaggcaaaatgccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 7980 tactcatact cttcctttttcaatattatt gaagcattta tcagggttat tgtctcatga 8040 gcggatacat atttgaatgtatttagaaaa ataaacaaat aggggttccg cgcacatttc 8100 cccgaaaagt gccacctgacgtctaagaaa ccattattat catgacatta acctataaaa 8160 ataggcgtat cacgaggccctttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct 8220 gacacatgca gctcccggagacggtcacag cttgtctgta agcggatgcc gggagcagac 8280 aagcccgtca gggcgcgtcagcgggtgttg gcgggtgtcg gggctggctt aactatgcgg 8340 catcagagca gattgtactgagagtgcacc atatgcggtg tgaaataccg cacagatgcg 8400 taaggagaaa ataccgcatcaggcgccatt cgccattcag gctgcgcaac tgttgggaag 8460 ggcgatcggt gcgggcctcttcgctattac gccagctggc gaaaggggga tgtgctgcaa 8520 ggcgattaag ttgggtaacgccagggtttt cccagtcacg acgttgtaaa acgacggcca 8580 gtgaattgga tttaggtgacactatagaat acgaattc 8618 6 20 DNA artificial syntheticoligodeoxynucleotide 1826 containing CpG motifs 6 tccatgacgt tcctgacgtt20 7 20 DNA artificial synthetic oligodeoxynucleotide 1982 7 tccaggacttctctcaggtt 20

What is claimed is:
 1. A composition comprising a recombinant MVA virus(rMVA) containing a nucleic acid sequence encoding p53.
 2. Thecomposition of claim 1, wherein said p53 is wild type murine p53.
 3. Thecomposition of claim 2, wherein said nucleic acid sequence comprises thenucleotide sequence of SEQ ID NO:
 1. 4. The composition of claim 1,wherein said p53 is wild type human p53.
 5. The composition of claim 4,wherein said nucleic acid sequence comprises the nucleotide sequence ofSEQ ID NO:
 2. 6. The composition of claim 1, further comprising animmunomodulator comprising a CTLA-4 blocking agent, a CpGoligodeoxynucleotide, or both a CTLA-4 blocking agent and a CpGoligodeoxynucleotide.
 7. The composition of claim 6, wherein said CTLA-4blocking agent is an antibody.
 8. The composition of claim 7, whereinsaid antibody is a monoclonal antibody.
 9. A method of treating asubject having a p53-expressing malignancy, comprising introducing intosaid subject a composition comprising recombinant MVA virus containing anucleic acid sequence encoding p53.
 10. The method of claim 9, whereinsaid subject is human.
 11. The method of claim 9, wherein introductionof said composition elicits an immune response effective against saidp53-expressing malignancy.
 12. The method of claim 9, wherein said p53is wild type human p53.
 13. The method of claim 12, wherein said nucleicacid sequence comprises the nucleotide sequence of SEQ ID NO:
 2. 14. Themethod of claim 9, wherein introduction of said composition is repeatedone or more times.
 15. The method of claim 9, further comprisingintroducing into said subject an immunomodulator comprising a CTLA-4blocking agent, a CpG oligodeoxynucleotide, or both a CTLA-4 blockingagent and a CpG oligodeoxynucleotide.
 16. The method of claim 15,wherein said CTLA-4 blocking agent is an antibody.
 17. The method ofclaim 16, wherein said antibody is a monoclonal antibody.
 18. The methodof claim 15, wherein introduction of said immunomodulator occurs priorto, simultaneous with, or after introduction of the compositioncomprising recombinant MVA virus.
 19. The method of claim 15, whereinintroduction of said immunomodulator is repeated one or more times. 20.The method of claims 9 or 15, wherein the method of said introduction isselected from the group consisting of subcutaneous, percutaneous,intradermal, intraperitoneal, intramuscular, intratumoral andintravenous injection.
 21. A kit for treating a subject having ap53-expressing malignancy comprising a composition comprisingrecombinant MVA virus containing a nucleic acid sequence encoding p53and instructions for administration of said composition, wherein saidadministration elicits an immune response effective against saidp53-expressing malignancy.
 22. The kit of claim 21, wherein said p53 ishuman p53.
 23. The kit of claim 22, wherein said nucleic acid sequencecomprises the nucleotide sequence of SEQ ID NO:
 2. 24. The kit of claim21, further comprising an immunomodulator comprising a CTLA4 blockingagent, a CpG oligodeoxynucleotide, or both a CTLA-4 blocking agent and aCpG oligodeoxynucleotide.
 25. The kit of claim 24, wherein said CTLA-4blocking agent is an antibody.
 26. The kit of claim 25, wherein saidantibody is a monoclonal antibody.
 27. The kit of claim 21, wherein saidsubject is human.
 28. A vector comprising an MVA recombination plasmidcontaining a nucleic acid insert encoding p53.
 29. The vector of claim28, wherein said MVA recombination plasmid is pLW22.
 30. The vector ofclaim 28, wherein said nucleic acid insert encodes human wild type p53.31. The vector of claim 30, wherein said vector comprises the sequenceof SEQ ID NO:
 5. 32. A method of generating a p53 specific cytotoxic Tlymphocyte (CTL) response against cells overexpressing mutant p53comprising administering a composition comprising recombinant MVA viruscontaining a nucleic acid sequence encoding p53.
 33. The method of claim32, wherein said nucleic acid sequence encoding p53 encodes murine wildtype p53.
 34. The method of claim 33, wherein said nucleic acid sequencecomprises the nucleotide sequence of SEQ ID NO:
 1. 35. The method ofclaim 32, wherein said nucleic acid sequence encoding p53 encodes humanwild type p53.
 36. The method of claim 35, wherein said nucleic acidsequence comprises the nucleotide sequence of SEQ ID NO:
 2. 37. Themethod of claim 32, further comprising administering an immunomodulatorcomprising a CTLA-4 blocking agent, a CpG oligodeoxynucleotide, or botha CTLA-4 blocking agent and a CpG oligodeoxynucleotide.
 38. The methodof claim 37, wherein said CTLA-4 blocking agent is an antibody.
 39. Themethod of claim 38, wherein said antibody is a monoclonal antibody. 40.The method of claim 37, wherein administration of said immunomodulatoroccurs prior to, simultaneous with, or after introduction of thecomposition comprising recombinant MVA virus.
 41. The method of claim37, wherein introduction of said immunomodulator is repeated one or moretimes.